U.S. patent application number 15/545302 was filed with the patent office on 2018-08-09 for methods, compositions, and systems for delivering therapeutic and diagnostic agents into cells.
The applicant listed for this patent is PHASERX, INC.. Invention is credited to Pierrot HARVIE, Michael E. HOUSTON, JR., Sean D. MONAHAN, Mary G. PRIEVE.
Application Number | 20180221402 15/545302 |
Document ID | / |
Family ID | 55310936 |
Filed Date | 2018-08-09 |
United States Patent
Application |
20180221402 |
Kind Code |
A1 |
PRIEVE; Mary G. ; et
al. |
August 9, 2018 |
METHODS, COMPOSITIONS, AND SYSTEMS FOR DELIVERING THERAPEUTIC AND
DIAGNOSTIC AGENTS INTO CELLS
Abstract
Disclosed are methods for delivering a therapeutic or diagnostic
agent to the cytosol of a cell in a subject. The disclosed methods
generally include administering to the subject an effective amount
of a lipid nanoparticle comprising the therapeutic or diagnostic
agent and an effective amount of a membrane-destabilizing polymer.
Also disclosed are related compositions and delivery systems.
Inventors: |
PRIEVE; Mary G.; (Lake
Forest Park, WA) ; HOUSTON, JR.; Michael E.;
(Kirkland, WA) ; HARVIE; Pierrot; (Seattle,
WA) ; MONAHAN; Sean D.; (Lake Forest Park,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHASERX, INC. |
Seattle |
WA |
US |
|
|
Family ID: |
55310936 |
Appl. No.: |
15/545302 |
Filed: |
January 21, 2016 |
PCT Filed: |
January 21, 2016 |
PCT NO: |
PCT/US2016/014232 |
371 Date: |
July 20, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62106024 |
Jan 21, 2015 |
|
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62173847 |
Jun 10, 2015 |
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62233568 |
Sep 28, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 47/32 20130101;
A61K 31/47 20130101; A61P 35/00 20180101; A61K 47/186 20130101;
A61K 9/1272 20130101; A61K 33/24 20130101; A61P 37/02 20180101;
A61K 31/7048 20130101; A61P 31/12 20180101; A61K 31/40 20130101;
A61K 31/475 20130101; A61K 31/407 20130101; A61K 9/5123 20130101;
A61K 31/5365 20130101; C08F 293/00 20130101; A61K 31/7105 20130101;
A61P 3/00 20180101; A61P 29/00 20180101; A61K 31/4745 20130101;
A61P 43/00 20180101; A61K 31/704 20130101; A61K 9/0019 20130101;
A61K 48/0041 20130101 |
International
Class: |
A61K 31/7105 20060101
A61K031/7105; A61K 47/32 20060101 A61K047/32; A61K 47/18 20060101
A61K047/18; A61K 48/00 20060101 A61K048/00; A61K 9/127 20060101
A61K009/127; A61K 9/51 20060101 A61K009/51; C08F 293/00 20060101
C08F293/00 |
Claims
1. A method for delivering a therapeutic or diagnostic agent to the
cytosol of a target cell within a subject, the method comprising:
administering to the subject (a) an effective amount of a lipid
nanoparticle comprising the therapeutic or diagnostic agent and (b)
an effective amount of a membrane-destabilizing polymer, wherein
the therapeutic or diagnostic agent is delivered to the cytosol of
the target cell.
2. The method of claim 1, wherein at least one of the lipid
nanoparticle and membrane-destabilizing polymer comprises a first
targeting ligand that specifically binds to a molecule on the
surface of the target cell.
3. The method of claim 1, wherein the lipid nanoparticle and
membrane-destabilizing polymer are administered separately.
4. The method of claim 3, wherein the membrane-destabilizing
polymer is administered after administration of the lipid
nanoparticle.
5. The method of claim 1, wherein the lipid nanoparticle and
membrane-destabilizing polymer are administered together within a
single composition.
6-16. (canceled)
17. The method claim 2, wherein the membrane-destabilizing polymer
comprises the first targeting ligand.
18. The method of claim 2, wherein the lipid nanoparticle comprises
the first targeting ligand.
19. The method of claim 2, wherein both the lipid nanoparticle and
the membrane-destabilizing polymer comprise the first targeting
ligand.
20-43. (canceled)
44. The method of claim 1, wherein the membrane-destabilizing
polymer is a pH-sensitive polymer.
45. The method of claim 44, wherein the pH-sensitive polymer is a
copolymer.
46-67. (canceled)
68. The method of claim 44, wherein the pH-sensitive
membrane-destabilizing polymer is a diblock copolymer having a
hydrophilic random copolymer block and a hydrophobic random
copolymer block, wherein the hydrophilic block is an amphiphilic
block comprising both hydrophilic monomeric residues and
hydrophobic monomeric residues, wherein the number of hydrophilic
monomeric residues in the hydrophilic block is greater than the
number of hydrophobic monomeric residues; wherein the hydrophobic
block is an amphiphilic, membrane-destabilizing block comprising
both hydrophobic monomeric residues and hydrophilic monomeric
residues and having an overall hydrophobic character at a pH of
about 7.4; and wherein each of the hydrophilic monomeric residues
of the hydrophilic and hydrophobic blocks is independently selected
from the group consisting of monomeric residues that are ionic at a
pH of about 7.4, monomeric residues that are neutral at a pH of
about 7.4, and monomeric residues that are zwitterionic at a pH of
about 7.4.
69. The method of claim 44, wherein the pH-sensitive polymer
comprises a random block copolymer of formula I: ##STR00041##
wherein A.sub.0, A.sub.1, A.sub.2, A.sub.3, A.sub.4 and A.sub.5 are
each independently selected from the group consisting of --C--C--,
--C(O)(C).sub.aC(O)O--, --O(C).sub.aC(O)--, --O(C).sub.b--, and
--CR.sub.8--CR.sub.9; wherein tetravalent carbon atoms of
A.sub.0-A.sub.5 that are not fully substituted with R.sub.1-R.sub.6
and Y.sub.0-Y.sub.5 are completed with an appropriate number of
hydrogen atoms; wherein a and b are each independently 1-4; and
wherein R.sub.8 and R.sub.9 are each independently selected from
the group consisting of --C(O)OH, --C(O)Oalkyl, and
--C(O)NR.sub.10, wherein R.sub.8 and R.sub.9 are optionally
covalently linked together to form a ring structure; Y.sub.5 is
hydrogen or is selected from the group consisting of
-(1C-10C)alkyl, -(3C-6C)cycloalkyl, --O-(1C-10C)alkyl,
--C(O)O(1C-10C)alkyl, --C(O)NR.sub.11 (1C-10C)alkyl, and
-(6C-10C)aryl, any of which is optionally substituted with one or
more fluorine atoms; Y.sub.0, Y.sub.3, and Y.sub.4 are each
independently selected from the group consisting of a covalent
bond, -(1C-10C)alkyl-, --C(O)O(2C-10C)alkyl-,
--OC(O)(1C-10C)alkyl-, --O(2C-10C)alkyl-, --S(2C-10C)alkyl-, and
--C(O)NR.sub.12(2C-10C)alkyl-; Y.sub.1 and Y.sub.2 are each
independently selected from the group consisting of a covalent
bond, -(1C-18C)alkyl-, -(3C-18C)branched alkyl,
--C(O)O(2C-18C)alkyl-, --C(O)O(2C-18C)branched alkyl,
--OC(O)(1C-18C)alkyl-, --OC(O)(1C-18C)branched alkyl-,
--O(2C-18C)alkyl-, --O(2C-18C)branched alkyl-, --S(2C-18C)alkyl-,
--S(2C-18C)branched alkyl-, --C(O)NR.sub.12(2C-18C)alkyl-, and
--C(O)NR.sub.12(2C-18C)branched alkyl-, wherein any alkyl or
branched alkyl group of Y1 or Y2 is optionally substituted with one
or more fluorine atoms; R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.8, R.sub.9, R.sub.10, R.sub.11, and
R.sub.12 are each independently hydrogen, --CN, or selected from
the group consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; Q.sub.0 is a residue
selected from the group consisting of residues which are
hydrophilic at physiologic pH; O--[(C).sub.2-3--O].sub.x--R.sub.7;
and O--[(C).sub.2-3--C(O)--NR.sub.13R.sub.14; wherein x is 1-48;
R.sub.7 is --CH.sub.3 or --CO.sub.2H; and R.sub.13 and R.sub.14 are
each independently hydrogen, --CN, or selected from the group
consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; Q.sub.1 and Q.sub.2
are each independently absent or selected from a residue which is
hydrophilic at normal physiological pH; a conjugatable or
functionalizable residue; a residue which is hydrophobic at normal
physiological pH; an alkyl group optionally substituted with one or
more fluorine atoms; and a branched alkyl group optionally
substituted with one or more fluorine atoms; Q.sub.3 is a residue
which is positively charged at normal physiological pH; Q.sub.4 is
a residue which is negatively charged at normal physiological pH,
but undergoes protonation at lower pH; m is a mole fraction of
greater than 0 to 1.0; n is a mole fraction of 0 to less than 1.0;
p is a mole fraction of 0 to less than 1.0; wherein m+n+p=1; q is a
mole fraction of 0.1 to 0.9; r is a mole fraction of 0.05 to 0.9; s
is present up to a mole fraction of 0.85; wherein q+r+s=1; v is
from 1 to 25 kDa; and w is from 1 to 50 kDa.
70-73. (canceled)
74. The method of claim 69, wherein the pH-sensitive polymer is a
polymer of formula II:
T1-L-[PEGMA.sub.m-PDSMA.sub.n-BPAM.sub.p].sub.v-[DMAEMA.sub.q-PAA.sub.r-B-
MA.sub.s].sub.w II wherein PEGMA is polyethyleneglycol methacrylate
residue with 2-20 ethylene glycol units; PDSMA is pyridyl disulfide
methacrylate residue; BPAM is 2-[2-Boc amino ethoxy] ethyl
methacrylate residue; BMA is butyl methacrylate residue; PAA is
propyl acrylic acid residue; DMAEMA is dimethylaminoethyl
methacrylate residue; m is a mole fraction of 0.6 to 1; n is a mole
fraction of 0 to 0.4; p is a mole fraction of 0 to 0.4; m+n+p=1; q
is a mole fraction of 0.2 to 0.75; r is a mole fraction of 0.05 to
0.6; s is a mole fraction of 0.2 to 0.75; q+r+s=1; v is 1 to 25
kDa; w is 1 to 25 kDa; T1 is absent or is a first targeting ligand;
and L is absent or is a linking moiety.
75. The method of claim 69, wherein the pH-sensitive polymer is a
polymer of formula V:
T1-L-[PEGMA.sub.m-M2.sub.n].sub.v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub.-
w V wherein PEGMA is polyethyleneglycol methacrylate residue with
2-20 ethylene glycol units; M2 is a methacrylate residue selected
from the group consisting of a (C4-C18)alkyl-methacrylate residue;
a (C4-C18)branched alkyl-methacrylate residue; a cholesteryl
methacrylate residue; a (C4-C18)alkyl-methacrylate residue
substituted with one or more fluorine atoms; and a (C4-C18)branched
alkyl-methacrylate residue substituted with one or more fluorine
atoms; BMA is butyl methacrylate residue; PAA is propyl acrylic
acid residue; DMAEMA is dimethylaminoethyl methacrylate residue; m
and n are each a mole fraction greater than 0, wherein m is greater
than n and m+n=1; q is a mole fraction of 0.2 to 0.75; r is a mole
fraction of 0.05 to 0.6; s is a mole fraction of 0.2 to 0.75;
q+r+s=1; v is 1 to 25 kDa; w is 1 to 25 kDa; T1 is absent or is the
first targeting ligand; and L is absent or is a linking moiety.
76-79. (canceled)
80. The method of claim 1, wherein the lipid nanoparticle comprises
the therapeutic agent.
81-82. (canceled)
83. The method of claim 80, wherein the therapeutic agent is the
polynucleotide.
84. The method of claim 83, wherein the lipid nanoparticle
comprises a mixture of lipid components comprising a cationic lipid
that is permanently charged at physiological pH, wherein said
cationic lipid is present in the mixture from about 35 mole % to
about 55 mole %; an ionizable anionic lipid, wherein said anionic
lipid is optionally absent and, if present, is present in the
mixture from about 25 mole % to about 40 mole %; a helper lipid,
wherein if the ionizable anionic lipid is absent, then the helper
lipid is present in the mixture from about 40 mole % to about 50
mole %, and if the ionizable anionic lipid is present, then the
helper lipid is present in the mixture from about 5 mole % to about
20 mole %; and a PEG-lipid, wherein the PEG-lipid is present in the
mixture from about 2 mole % to about 15 mole %.
85-90. (canceled)
91. The method of claim 83, wherein the polynucleotide is an
mRNA.
92. The method of claim 91, wherein the mRNA encodes a functional
protein associated with a protein deficiency disease.
93-114. (canceled)
115. A composition for delivering a therapeutic or diagnostic agent
to the cytosol of a target cell within a subject, the composition
comprising: a lipid nanoparticle comprising the therapeutic or
diagnostic agent, and a membrane-destabilizing polymer.
116-225. (canceled)
226. A pH-sensitive, membrane-destabilizing polymer comprising a
random block copolymer of formula Ia: ##STR00042## wherein A.sub.0,
A.sub.1, A.sub.2, A.sub.3, A.sub.4, A.sub.5 are each independently
selected from the group consisting of --C--C--,
--C(O)(C).sub.aC(O)O--, --O(C).sub.aC(O)--, --O(C).sub.b--, and
--CR.sub.8--CR.sub.9--; wherein tetravalent carbon atoms of
A.sub.0-A.sub.5 that are not fully substituted with R1-R6 and Y0-Y5
are completed with an appropriate number of hydrogen atoms; wherein
a and bare each independently 1-4; and wherein R.sub.8 and R.sub.9
are each independently selected from the group consisting of
--C(O)OH, --C(O)Oalkyl, and --C(O)NR.sub.10, wherein R.sub.8 and
R.sub.9 are optionally covalently linked together to form a ring
structure; Y.sub.5 is hydrogen or is selected from the group
consisting of -(1C-10C)alkyl, -(3C-6C)cycloalkyl,
--O-(1C-10C)alkyl, --C(O)O(1C-10C)alkyl, --C(O)NR.sub.11
(1C-10C)alkyl, and -(6C-10C)aryl, any of which is optionally
substituted with one or more fluorine atoms; Y.sub.0, Y.sub.3, and
Y.sub.4 are each independently selected from the group consisting
of a covalent bond, -(1C-10C)alkyl-, --C(O)O(2C-10C)alkyl-,
--OC(O)(1C-10C)alkyl-, --O(2C-10C)alkyl-, --S(2C-10C)alkyl-, and
--C(O)NR.sub.12(2C-10C) alkyl-; Y.sub.1 and Y.sub.2 are each
independently selected from the group consisting of a covalent
bond, -(1C-18C)alkyl-, -(3C-18C)branched alkyl,
--C(O)O(2C-18C)alkyl-, --C(O)O(2C-18C)branched alkyl,
--OC(O)(1C-18C)alkyl-, --OC(O)(1C-18C)branched alkyl-,
--O(2C-18C)alkyl-, --O(2C-18C)branched alkyl-, --S(2C-18C)alkyl-,
--S(2C-18C)branched alkyl-, --C(O)NR.sub.12(2C-18C)alkyl-, and
--C(O)NR.sub.12(2C-18C)branched alkyl-, wherein any alkyl or
branched alkyl group of Y1 or Y2 is optionally substituted with one
or more fluorine atoms; R.sub.1, R.sub.2, R.sub.3, R.sub.4,
R.sub.5, R.sub.6, R.sub.8, R.sub.9, R.sub.10, R.sub.11, and
R.sub.12 are each independently hydrogen, --CN, or selected from
the group consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; Q.sub.0 is a residue
selected from the group consisting of residues which are
hydrophilic at physiologic pH; O--[(C).sub.2-3--O].sub.x--R.sub.7;
and O--[(C).sub.2-3O]x-C(O)--NR.sub.13R.sub.14; wherein x is 1-48;
R.sub.7 is --CH.sub.3 or --CO.sub.2H; and R.sub.13 and R.sub.14 are
each independently hydrogen, --CN, or selected from the group
consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; Q.sub.1 and Q.sub.2
are each independently absent or selected from a residue which is
hydrophilic at normal physiological pH; a conjugatable or
functionalizable residue; a residue which is hydrophobic at normal
physiological pH; an alkyl group optionally substituted with one or
more fluorine atoms; and a branched alkyl group optionally
substituted with one or more fluorine atoms; Q.sub.3 is a residue
which is positively charged at normal physiological pH; Q.sub.4 is
a residue which is negatively charged at normal physiological pH,
but undergoes protonation at lower pH; m is a mole fraction of
greater than 0.5 to less than 1.0; n is a mole fraction of greater
than 0 to less than 0.5; p is a mole fraction of 0 to less than
0.5; wherein m+n+p=1; q is a mole fraction of 0.1 to 0.9; r is a
mole fraction of 0.05 to 0.9; s is present up to a mole fraction of
0.85; wherein q+r+s=1; v is from 1 to 25 kDa; w is from 1 to 50
kDa; and at least one of Y.sub.1 and Q.sub.1 contains the alkyl or
branched alkyl group substituted with the one or more fluorine
atoms.
227-238. (canceled)
239. A pH-sensitive, membrane-destabilizing polymer of formula V:
T1-L-[PEGMA.sub.m-M2.sub.n].sub.v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub.-
w V wherein PEGMA is polyethyleneglycol methacrylate residue with
2-20 ethylene glycol units; M2 is a methacrylate residue selected
from the group consisting of a (C4-C18)alkyl-methacrylate residue;
a (C4-C18)branched alkyl-methacrylate residue; a cholesteryl
methacrylate residue; a (C4-C18)alkyl-methacrylate residue
substituted with one or more fluorine atoms; and a (C4-C18)branched
alkyl-methacrylate residue substituted with one or more fluorine
atoms; BMA is butyl methacrylate residue; PAA is propyl acrylic
acid residue; DMAEMA is dimethylaminoethyl methacrylate residue; m
and n are each a mole fraction greater than 0, wherein m is greater
than n and m+n=1; q is a mole fraction of 0.2 to 0.75; r is a mole
fraction of 0.05 to 0.6; s is a mole fraction of 0.2 to 0.75;
q+r+s=1; v is 1 to 25 kDa; w is 1 to 25 kDa; T1 is absent or is the
first targeting ligand; and L is absent or is a linking moiety.
240. (canceled)
241. A lipid nanoparticle comprising: (a) a polynucleotide; and (b)
a mixture of lipid components comprising a cationic lipid that is
permanently charged at physiological pH, wherein said cationic
lipid is present in the mixture from about 35 mole % to about 55
mole %; an ionizable anionic lipid, wherein said anionic lipid is
optionally absent and, if present, is present in the mixture from
about 25 mole % to about 40 mole %; a helper lipid, wherein if the
ionizable anionic lipid is absent, then the helper lipid is present
in the mixture from about 40 mole % to about 50 mole %, and if the
ionizable anionic lipid is present, then the helper lipid is
present in the mixture from about 5 mole % to about 20 mole %; and
a PEG-lipid, wherein the PEG-lipid is present in the mixture from
about 5 mole % to about 15 mole %.
242-246. (canceled)
247. A method for treating a disease characterized by a genetic
defect that results in a deficiency of a functional protein, the
method comprising: administering to a subject having the disease
(a) an effective amount of a lipid nanoparticle comprising an mRNA
that encodes the functional protein or a protein having the same
biological activity as the functional protein and (b) an effective
amount of a membrane-destabilizing polymer, wherein the mRNA is
delivered to the cytosol of target cells of a target tissue
associated with the disease, and wherein the mRNA is translated
during protein synthesis so as to produce the encoded protein
within the target tissue, thereby treating the disease.
248-272. (canceled)
Description
[0001] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII Copy, created
on Jan. 13, 2016, is named "3900_PCT1_Seq_Listing_ST25" and is
66,448 bytes in size.
BACKGROUND OF THE INVENTION
[0002] Lipid nanoparticles (LNPs) are effective drug delivery
systems for biologically active compounds such as therapeutic
nucleic acids, proteins, and peptides, which are otherwise cell
impermeable. Liposomal formulations have also been developed for
small molecule drugs, generally with the aim to enrich the drug in
certain tissues as well as to mitigate toxicity.
[0003] Drugs based on nucleic acids, which include large nucleic
acid molecules such as, e.g., in vitro transcribed messenger RNA
(mRNA) as well as smaller polynucleotides that interact with a
messenger RNA or a gene, have to be delivered to the proper
cellular compartment in order to be effective. For example,
double-stranded nucleic acids such as double-stranded RNA molecules
(dsRNA), including, e.g., siRNAs, suffer from their
physico-chemical properties that render them impermeable to cells.
Upon delivery into the proper compartment, siRNAs block gene
expression through a highly conserved regulatory mechanism known as
RNA interference (RNAi). Typically, siRNAs are large in size with a
molecular weight ranging from 12-17 kDa, and are highly anionic due
to their phosphate backbone with up to 50 negative charges. In
addition, the two complementary RNA strands result in a rigid
helix. These features contribute to the siRNA's poor "drug-like"
properties. When administered intravenously, the siRNA is rapidly
excreted from the body with a typical half-life in the range of
only 10 minutes. Additionally, siRNAs are rapidly degraded by
nucleases present in blood and other fluids or in tissues, and have
been shown to stimulate strong immune responses in vitro and in
vivo. See, e.g., Robbins et al., Oligonucleotides 19:89-102, 2009.
mRNA molecules suffer from similar issues of impermeability,
fragility, and immunogenicity.
[0004] By introduction of appropriate chemical modifications,
stability towards nucleases can be increased and at the same time
immune stimulation can be suppressed. Conjugation of lipophilic
small molecules to the siRNAs improves the pharmacokinetic
characteristics of the double-stranded RNA molecule. It has been
demonstrated that these small molecule siRNA conjugates are
efficacious in a specific down regulation of a gene expressed in
hepatocytes of rodents. However, in order to elicit the desired
biologic effect, a large dose was needed. See Soutschek et al.,
Nature 432:173-178, 2004.
[0005] Lipid nanoparticle formulations have improved nucleic acid
delivery in vivo. For example, such formulations have significantly
reduced siRNA doses necessary to achieve target knockdown in vivo.
See Zimmermann et al., Nature 441:111-114, 2006. Typically, such
lipid nanoparticle drug delivery systems are multi-component
formulations comprising cationic lipids, helper lipids, and lipids
containing polyethylene glycol. The positively charged cationic
lipids bind to the anionic nucleic acid, while the other components
support a stable self-assembly of the lipid nanoparticles.
[0006] Efforts have been directed toward improving delivery
efficacy of lipid nanoparticle formulations. Many such efforts have
been aimed toward developing more appropriate cationic lipids. See,
e.g., Akinc et al., Nature Biotechnology 26:561-569, 2008; Love et
al., Proc. Natl. Acad. Sci. USA 107:1864-1869, 2010; Baigude et
al., Journal of Controlled Release 107:276-287, 2005; Semple et
al., Nature Biotechnology 28:172-176, 2010. Despites these efforts,
improvements in terms of increased efficacy and/or decreased
toxicity are still needed, especially for lipid nanoparticle based
drug delivery systems intended for therapeutic uses.
SUMMARY OF THE INVENTION
[0007] In one aspect, the present invention provides a method for
delivering a therapeutic or diagnostic agent to the cytosol of a
target cell within a subject. The method generally includes
administering to the subject (a) an effective amount of a lipid
nanoparticle comprising the therapeutic or diagnostic agent and (b)
an effective amount of a membrane-destabilizing polymer, where the
therapeutic or diagnostic agent is delivered to the cytosol of the
target cell. The lipid nanoparticle and membrane-destabilizing
polymer can be administered separately (e.g., the
membrane-destabilizing polymer administered after administration of
the lipid nanoparticle) or, alternatively, together within a single
composition. Typically, the lipid nanoparticle is less than about
200 nm in size. In certain variations, the lipid nanoparticle and
the membrane-destabilizing polymer are administered in a repeat
dosage regime (e.g., a weekly or bi-weekly repeated administration
protocol).
[0008] In some embodiments, the lipid nanoparticle comprises a
cationic lipid. Particularly suitable cationic lipids include
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA); N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
chloride (DOTAP); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(DOEPC); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC);
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC);
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1),
N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarb-
oxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5);
Dioctadecylamido-glycylspermine (DOGS);
3b-[N--(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol);
Dioctadecyldimethylammonium Bromide (DDAB); a Saint lipid (e.g.,
SAINT-2, N-methyl-4-(dioleyl)methylpyridinium);
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide
(DMRIE); 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide
(DORIE); 1,2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium
chloride (DORI); Di-alkylated Amino Acid (DILA.sup.2) (e.g.,
C18:1-norArg-C16); Dioleyldimethylammonium chloride (DODAC);
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC); and
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC). In
some variations, the cationic lipid is an ionizable cationic lipid
such as, e.g., Dioctadecyldimethylammonium bromide (DDAB),
1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA),
2,2-dilinoleyl-4-(2dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (DLin-MC3-DMA),
1,2-Dioleoyloxy-3-dimethylaminopropane (DODAP),
1,2-Dioleyloxy-3-dimethylaminopropane (DODMA),
Morpholinocholesterol (Mo-CHOL),
(R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride
(DODAPen-C1), (R)-5-guanidinopentane-1,2-diyl dioleate
hydrochloride (DOPen-G),
(R)--N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride
(DOTAPen). In certain embodiments, a lipid nanoparticle includes a
combination or two or more cationic lipids (e.g., two or more
cationic lipids as above).
[0009] In some embodiments of a method as above, the lipid
nanoparticle includes an ionizable anionic lipid such as, e.g.,
cholesteryl hemisuccinate (CHEMS), phosphatidylserine,
palmitoylhomoserine, or .alpha.-tocopherol hemisuccinate. In
certain variations, a lipid nanoparticle includes a combination or
two or more ionizable anionic lipids (e.g., two or more ionizable
anionic lipids as above).
[0010] In some variations of a method as above, the lipid
nanoparticle includes a helper lipid. Particularly suitable helper
lipids includes cholesterol (CHOL);
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC);
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC);
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC);
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE);
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE);
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE); and
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE). In
certain embodiments, a lipid nanoparticle includes a combination or
two or more helper lipids (e.g., two or more helper lipids as
above).
[0011] In certain embodiments of a method as above, the lipid
nanoparticle includes a polyethylenegycol-lipid conjugate
(PEG-lipid) such as, e.g.,
N-(Carbonyl-methoxypolyethyleneglycol.sub.n)-1,2-dimyristoyl-sn-glycero-3-
-phosphoethanolamine (DMPE-PEG.sub.n where n is 350, 500, 750, 1000
or 2000),
N-(Carbonyl-methoxypolyethyleneglycol.sub.n)-1,2-distearoyl-sn-gly-
cero-3-phosphoethanolamine (DSPE-PEG.sub.n where n is 350, 500,
750, 1000 or 2000), DSPE-polyglycelin-cyclohexyl-carboxylic acid,
DSPE-polyglycelin-2-methylglutar-carboxylic acid, polyethylene
glycol-dimyristolglycerol (PEG-DMG), polyethylene glycol-distearoyl
glycerol (PEG-DSG), or
N-octanoyl-sphingosine-1-{(succinyl[methoxy(polyethylene
glycol)2000]} (C8 PEG2000 Ceramide). In some variations of
DMPE-PEG.sub.n where n is 350, 500, 750, 1000 or 2000, the
PEG-lipid is N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG
2,000). In some variations of DSPE-PEG.sub.n where n is 350, 500,
750, 1000 or 2000, the PEG-lipid is
N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG
2,000). In certain embodiments, a lipid nanoparticle includes a
combination or two or more PEG-lipids (e.g., two or more PEG-lipids
as above).
[0012] In some embodiments of a method as above, at least one of
the lipid nanoparticle and membrane-destabilizing polymer includes
a first targeting ligand that specifically binds to a molecule on
the surface of the target cell. The membrane-destabilizing polymer,
the lipid nanoparticle, or both the membrane-destabilizing polymer
and lipid nanoparticle may include the first targeting ligand. In
some embodiments, one of the lipid nanoparticle and
membrane-destabilizing polymer includes the first targeting ligand,
and the other of the lipid nanoparticle and membrane-destabilizing
polymer includes a second targeting ligand that is different from
the first targeting ligand and either (i) specifically binds to the
same cell surface molecule recognized by the first targeting ligand
or (ii) specifically binds to a different cell surface molecule on
the surface of the target cell. In particular variations, either
the first targeting ligand, the second targeting ligand, or both
the first and second targeting ligands specifically bind(s) to a
cell surface molecule selected from transferrin receptor type 1,
transferrin receptor type 2, the EGF receptor, HER2/Neu, a VEGF
receptor, a PDGF receptor, an integrin, an NGF receptor, CD2, CD3,
CD4, CD8, CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69, the
asialoglycoprotein receptor (ASGPR), prostate-specific membrane
antigen (PSMA), a folate receptor, and a sigma receptor.
[0013] In certain embodiments of a method as above in which at
least one of the lipid nanoparticle and membrane-destabilizing
polymer includes a first targeting ligand (and the other of the
lipid nanoparticle and membrane-destabilizing polymer optionally
includes a second targeting ligand), the first and/or second
targeting ligand includes a small molecule targeting moiety. In
specific variations, the small molecule targeting moiety is a sugar
(e.g., lactose, galactose, N-acetyl galactosamine (NAG, also
referred to as GalNAc), mannose, and mannose-6-phosphate (M6P)), a
vitamin (e.g., folate), a bisphosphonate, or an analogue thereof.
In other embodiments, the first and/or second targeting ligand is a
protein such as, e.g., an antibody, a peptide aptamer, or a protein
derived from a natural ligand of the cell surface molecule. In yet
other embodiments, the first and/or second targeting ligand is a
peptide such as, e.g., an integrin-binding peptide, a LOX-1-binding
peptide, and epidermal growth factor (EGF) peptide, a neurotensin
peptide, an NL4 peptide, or a YIGSR laminin peptide.
[0014] In certain embodiments of a method as above, target cell is
selected from a secretory cell, a chondrocyte, an epithelial cell,
a nerve cell, a muscle cell, a blood cell, an endothelial cell, a
pericyte, a fibroblast, a glial cell, and a dendritic cell. Other
suitable target cells include cancer cells, immune cells,
bacterially-infected cells, virally-infected cells, or cells having
an abnormal metabolic activity.
[0015] In a particular variation where the target cell is a
secretory cell, the target secretory cell is a hepatocyte. In some
such embodiments, at least one of the lipid nanoparticle and
membrane-destabilizing polymer includes a first targeting ligand
that specifically binds to a molecule on the surface of the
hepatocyte. In certain embodiments, the first targeting ligand
specifically binds to the asialoglycoprotein receptor (ASGPR); for
example, in particular variations, the first targeting ligand
includes an N-acetylgalactosamine (NAG) residue. In some
embodiments as above comprising a first targeting ligand that binds
to a molecule on the surface of hepatocytes, both the lipid
nanoparticle and the membrane-destabilizing polymer include the
first targeting ligand. In other embodiments one of the lipid
nanoparticle and membrane-destabilizing polymer includes the first
targeting ligand, and the other of the lipid nanoparticle and
membrane destabilizing polymer includes a second targeting ligand
that is different from the first targeting ligand and either (i)
specifically binds to the asialoglycoprotein receptor (ASGPR) or
(ii) specifically binds to a different cell surface molecule on the
surface of the hepatocyte; in some such embodiments, the second
targeting ligand includes an N-acetylgalactosamine (NAG)
residue.
[0016] In some embodiments of a method as above, the
membrane-destabilizing polymer is a copolymer, a synthetic peptide,
a membrane-destabilizing toxin or derivative thereof, or a viral
fusogenic peptide or derivative thereof. In a particular variation,
the membrane-destabilizing polymer is a pH-sensitive polymer such
as, e.g., a pH-sensitive copolymer. The copolymer may be a block
copolymer such as, for example, a diblock copolymer. In some
variations, the block copolymer includes a hydrophobic,
membrane-destabilizing block and a hydrophilic block. In some such
embodiments, the hydrophilic block is polymerized from both
hydrophilic monomers and hydrophobic monomers such that there are
more hydrophilic monomeric residues than hydrophobic monomeric
residues in the hydrophilic block. The hydrophilic block may be
cleavably linked to the hydrophobic block, such as through a
disulfide bond or a pH-sensitive bond. In some embodiments, the
hydrophilic block includes monomeric residues linked to a pendant
shielding moiety such as, e.g., a polyethylene glycol (PEG) moiety.
The shielding moiety may be cleavably linked to the hydrophilic
block, such as through a disulfide bond or a pH-sensitive bond.
Particularly suitable pH-sensitive bonds (for linkage of the
hydrophilic and hydrophobic blocks or linkage of the shielding
moiety to the hydrophilic block) include hydrazone, acetal, ketal,
imine, orthoester, carbonate, and maleamic acid linkages.
[0017] The pH-sensitive polymer may include monomeric residues
having a carboxylic acid functional group, monomeric residues
having an amine functional group, and/or monomeric residues having
a hydrophobic functional group. In some variations, the
pH-sensitive polymer includes monomeric residues derived from
polymerization of a (C.sub.2-C.sub.8) alkylacrylic acid (e.g.,
propylacrylic acid); monomeric residues derived from polymerization
of a (C.sub.2-C.sub.8) alkyl-ethacrylate, a (C.sub.2-C.sub.8)
alkyl-methacrylate, or a (C.sub.2-C.sub.8) alkyl-acrylate; and/or
monomeric residues derived from polymerization of
(N,N-di(C.sub.1-C.sub.6)alkyl-amino(C.sub.1-C.sub.6)alkyl-ethacrylate,
(N,N-di(C.sub.1-C.sub.6)alkyl-amino(C.sub.1-C.sub.6)alkyl-methacrylate,
or
(N,N-di(C.sub.1-C.sub.6)alkyl-amino(C.sub.1-C.sub.6)alkyl-acrylate.
In a specific variation, the pH-sensitive polymer includes a random
copolymer chain having monomeric residues derived from
polymerization of propyl acrylic acid,
N,N-dimethylaminoethylmethacrylate, and butyl methacrylate; in some
such embodiments, the pH-sensitive polymer is a block copolymer
comprising the random copolymer chain as a membrane disrupting
polymer block, and further including one or more additional
blocks.
[0018] In certain embodiments, the pH-sensitive
membrane-destabilizing polymer is a diblock copolymer having a
hydrophilic random copolymer block and a hydrophobic random
copolymer block, where (i) the hydrophilic block is an amphiphilic
block comprising both hydrophilic monomeric residues and
hydrophobic monomeric residues, where the number of hydrophilic
monomeric residues in the hydrophilic block is greater than the
number of hydrophobic monomeric residues, (ii) the hydrophobic
block is an amphiphilic, membrane-destabilizing block comprising
both hydrophobic monomeric residues and hydrophilic monomeric
residues and having an overall hydrophobic character at a pH of
about 7.4; and (iii) each of the hydrophilic monomeric residues of
the hydrophilic and hydrophobic blocks is independently selected
from the group consisting of monomeric residues that are ionic at a
pH of about 7.4, monomeric residues that are neutral at a pH of
about 7.4, and monomeric residues that are zwitterionic at a pH of
about 7.4.
[0019] In yet other variations, the pH-sensitive polymer is
covalently linked to a membrane-destabilizing peptide. In some such
embodiments, the pH-sensitive polymer includes a plurality of
pendant linking groups, and a plurality of membrane-destabilizing
peptides are linked to the pH-sensitive polymer via the plurality
of pendant linking groups.
[0020] In some embodiments, the pH-sensitive polymer includes a
random block copolymer of formula I:
##STR00001## [0021] where [0022] A.sub.0, A.sub.1, A.sub.2,
A.sub.3, A.sub.4 and A.sub.5 are each independently selected from
the group consisting of --C--C--, --C(O)(C).sub.aC(O)O--,
--O(C).sub.aC(O)--, --O(C).sub.b--, and --CR.sub.8--CR.sub.9; where
tetravalent carbon atoms of A.sub.0-A.sub.5 that are not fully
substituted with R.sub.1-R.sub.6 and Y.sub.0-Y.sub.5 are completed
with an appropriate number of hydrogen atoms; wherein a and b are
each independently 1-4; and where R.sub.8 and R.sub.9 are each
independently selected from the group consisting of --C(O)OH,
--C(O)Oalkyl, and --C(O)NR.sub.10, where R.sub.8 and R.sub.9 are
optionally covalently linked together to form a ring structure
(e.g., a cyclic anhydride or cyclic imide); [0023] Y.sub.5 is
hydrogen or is selected from the group consisting of
-(1C-10C)alkyl, -(3C-6C)cycloalkyl, --O-(1C-10C)alkyl,
--C(O)O(1C-10C)alkyl, --C(O)NR.sub.11(1C-10C)alkyl, and
-(6C-10C)aryl, any of which is optionally substituted with one or
more fluorine atoms; [0024] Y.sub.0, Y.sub.3, and Y.sub.4 are each
independently selected from the group consisting of a covalent
bond, -(1C-10C)alkyl-, --C(O)O(2C-10C)alkyl-,
--OC(O)(1C-10C)alkyl-, --O(2C-10C)alkyl-, --S(2C-10C)alkyl-, and
--C(O)NR.sub.12(2C-10C)alkyl-; [0025] Y.sub.1 and Y.sub.2 are each
independently selected from the group consisting of a covalent
bond, -(1C-18C)alkyl-, -(3C-18C)branched alkyl,
--C(O)O(2C-18C)alkyl-, --C(O)O(2C-18C)branched alkyl,
--OC(O)(1C-18C)alkyl-, --OC(O)(1C-18C)branched alkyl-,
--O(2C-18C)alkyl-, --O(2C-18C)branched alkyl-, --S(2C-18C)alkyl-,
--S(2C-18C)branched alkyl-, --C(O)NR.sub.12(2C-18C)alkyl-, and
--C(O)NR.sub.12(2C-18C)branched alkyl-, where any alkyl or branched
alkyl group of Y.sub.1 or Y.sub.2 is optionally substituted with
one or more fluorine atoms; [0026] R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.8, R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently hydrogen, --CN, or selected
from the group consisting of alkyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl and heteroaryl, any of which is
optionally substituted with one or more fluorine atoms; [0027]
Q.sub.0 is a residue selected from the group consisting of residues
which are hydrophilic at physiologic pH;
O--[(C).sub.2-3--O].sub.x--R.sub.7; and
O--[(C).sub.2-3--O].sub.x--C(O)--NR.sub.13R.sub.14; where x is
1-48; R.sub.7 is --CH.sub.3 or --CO.sub.2H; and R.sub.13 and
R.sub.14 are each independently hydrogen, --CN, or selected from
the group consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; [0028] Q.sub.1 and
Q.sub.2 are each independently absent or selected from a residue
which is hydrophilic at normal physiological pH; a conjugatable or
functionalizable residue; a residue which is hydrophobic at normal
physiological pH; an alkyl group optionally substituted with one or
more fluorine atoms; and a branched alkyl group optionally
substituted with one or more fluorine atoms; [0029] Q.sub.3 is a
residue which is positively charged at normal physiological pH;
[0030] Q.sub.4 is a residue which is negatively charged at normal
physiological pH, but undergoes protonation at lower pH; [0031] m
is a mole fraction of greater than 0 to 1.0; [0032] n is a mole
fraction of 0 to less than 1.0; [0033] p is a mole fraction of 0 to
less than 1.0; wherein m+n+p=1; [0034] q is a mole fraction of 0.1
to 0.9; [0035] r is a mole fraction of 0.05 to 0.9; [0036] s is
present up to a mole fraction of 0.85; wherein q+r+s=1; [0037] v is
from 1 to 25 kDa; and [0038] w is from 1 to 50 kDa.
[0039] In some embodiments comprising a pH-sensitive polymer of
formula I as above, m is greater than n+p. In some such variations,
p is 0.
[0040] In some embodiments comprising a pH-sensitive polymer of
formula I as above, n is greater than 0. In some such variations,
at least one of Y.sub.1 and Q.sub.1 contains the alkyl or branched
alkyl group substituted with the one or more fluorine atoms. In
more particular variations, p is 0 and/or m is greater than n.
[0041] In certain embodiments comprising a pH-sensitive polymer of
formula I, the pH-sensitive polymer is a polymer of formula II:
T1-L-[PEGMA.sub.m-PDSMA.sub.n-BPAM.sub.p]v-[DMAEMA.sub.q-PAA.sub.r-BMA.s-
ub.s].sub.w II [0042] where [0043] PEGMA is polyethyleneglycol
methacrylate residue with 2-20 ethylene glycol units; [0044] PDSMA
is pyridyl disulfide methacrylate residue; [0045] BPAM is 2-[2-Boc
amino ethoxy] ethyl methacrylate residue; [0046] BMA is butyl
methacrylate residue; [0047] PAA is propyl acrylic acid residue;
[0048] DMAEMA is dimethylaminoethyl methacrylate residue; [0049] m
is a mole fraction of 0.6 to 1; [0050] n is a mole fraction of 0 to
0.4 (e.g., 0 to 0.2); [0051] p is a mole fraction of 0 to 0.4
(e.g., 0 to 0.2); [0052] m+n+p=1; [0053] q is a mole fraction of
0.2 to 0.75; [0054] r is a mole fraction of 0.05 to 0.6; [0055] s
is a mole fraction of 0.2 to 0.75; [0056] q+r+s=1; [0057] v is 1 to
25 kDa; [0058] w is 1 to 25 kDa; [0059] T1 is absent or is the
first targeting ligand; and [0060] L is absent or is a linking
moiety.
[0061] In other embodiments comprising a pH-sensitive polymer of
formula I, the pH-sensitive polymer is a polymer of formula V:
T1-L-[PEGMA.sub.m-M2.sub.n].sub.v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub-
.w V [0062] where [0063] PEGMA is polyethyleneglycol methacrylate
residue with 2-20 ethylene glycol units; [0064] M2 is a
methacrylate residue selected from the group consisting of [0065] a
(C4-C18)alkyl-methacrylate residue; [0066] a (C4-C18)branched
alkyl-methacrylate residue; [0067] a cholesteryl methacrylate
residue; [0068] a (C4-C18)alkyl-methacrylate residue substituted
with one or more fluorine atoms; and [0069] a (C4-C18)branched
alkyl-methacrylate residue substituted with one or more fluorine
atoms; [0070] BMA is butyl methacrylate residue; [0071] PAA is
propyl acrylic acid residue; [0072] DMAEMA is dimethylaminoethyl
methacrylate residue; [0073] m and n are each a mole fraction
greater than 0, wherein m is greater than n and m+n=1; [0074] q is
a mole fraction of 0.2 to 0.75; [0075] r is a mole fraction of 0.05
to 0.6; [0076] s is a mole fraction of 0.2 to 0.75; [0077] q+r+s=1;
[0078] v is 1 to 25 kDa; [0079] w is 1 to 25 kDa; [0080] T1 is
absent or is the first targeting ligand; and [0081] L is absent or
is a linking moiety.
[0082] In some specific embodiments of a polymer of formula V, M2
is selected from 2,2,3,3,4,4,4-heptafluorobutyl methacrylate
residue; 3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl
methacrylate residue;
2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl 2-methylacrylate
residue; 3,3,4,4,5,5,6,6,6-nonafluorohexyl methacrylate residue
(also referred to as 2-propenoic acid, 2-methyl-,
3,3,4,4,5,5,6,6,6-nonafluorohexyl ester residue);
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue;
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue; 2-ethylhexyl methacrylate residue; butyl
methacrylate residue; hexyl methacrylate residue; octyl
methacrylate residue; n-decyl methacrylate residue; lauryl
methacrylate residue; myristyl methacrylate residue; stearyl
methacrylate residue; cholesteryl methacrylate residue; ethylene
glycol phenyl ether methacrylate residue; 2-propenoic acid,
2-methyl-, 2-phenylethyl ester residue; 2-propenoic acid,
2-methyl-, 2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl ester
residue; 2-propenoic acid, 2-methyl-, 2-(1H-imidazol-1-yl)ethyl
ester residue; 2-propenoic acid, 2-methyl-, cyclohexyl ester
residue; 2-propenoic acid, 2-methyl-,
2-[bis(1-methylethyl)amino]ethyl ester residue; 2-propenoic acid,
2-methyl-, 3-methylbutyl ester residue; neopentyl methacrylate
residue; tert-butyl methacrylate residue; 3,3,5-trimethyl
cyclohexyl methacrylate residue; 2-hydroxypropyl methacrylate
residue; 5-nonyl methacrylate residue; 2-butyl-1-octyl methacrylate
residue; 2-hexyl-1-decyl methacrylate residue; and 2-(tert-butyl
amino)ethyl methacrylate residue.
[0083] In particular variations of a method as above comprising a
pH-sensitive polymer of formula II or formula V, PEGMA has 4-5
ethylene glycol units or 7-8 ethylene glycol units; T1 and L are
present and T1 includes an N-acetylgalactosamine (NAG) residue;
and/or L includes a polyethylene glycol (PEG) moiety having 2-20
ethylene glycol units.
[0084] In certain embodiments, the lipid nanoparticle includes the
therapeutic agent. The therapeutic agent may be an anti-cancer
agent, an anti-viral agent, an immunomodulatory agent, an
anti-inflammatory agent, or an agent that modulates a cellular
metabolic activity. Suitable therapeutic agents may be selected
from polynucleotides, proteins, peptides, and small molecules.
[0085] In some embodiments, the therapeutic agent is a
polynucleotide. In some such variations, the lipid nanoparticle has
an N:P (nitrogen to phosphate) ratio of about 1 to about 30. In
certain embodiments, the polynucleotide is an mRNA, such as, for
example, an mRNA encoding a functional protein associated with a
protein deficiency disease. In particular variations, the target
cell is a hepatocyte and the mRNA encodes a protein selected from
the group consisting of alpha-1-antitrypsin (A1AT), carbamoyl
phosphate synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme,
alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase
(MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl
CoA carboxylase beta subunit (PCCB), a subunit of branched-chain
ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC),
copper-transporting ATPase Atp7B, bilirubin uridinediphosphate
glucuronyltransferase (BGT) enzyme, hepcidin, glucose-6-phosphatase
(G6Pase), glucose 6-phosphate translocase, lysosomal
glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1),
Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor
IX, galactose-1-phosphate uridylyltransferase, galactokinase,
UDP-galactose 4-epimerase, transthyretin, a complement regulatory
protein, phenylalanine hydroxylase (PAH), homogentisate
1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine
phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL),
argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1,
alpha-galactosidase A, acid ceramidase, acid .alpha.-L-fucosidase,
acid f-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase,
galactocerebrosidase, acid .alpha.-mannosidase, .beta.-mannosidase,
arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate
sulfatase, acid f-galactosidase, acid .alpha.-glucosidase,
.beta.-hexosaminidase B, heparan-N-sulfatase,
alpha-N-acetylglucosaminidase, acetyl-CoA:.alpha.-glucosaminide
N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase,
alpha-N-acetylgalactosaminidase, sialidase, .beta.-glucuronidase,
.beta.-hexosaminidase A. In some embodiments, the polynucleotide is
a DNA, such as, for example, a DNA encoding a functional protein
associated with a protein deficiency disease (e.g., a protein
selected from the proteins listed above).
[0086] In certain embodiments, the therapeutic agent is an mRNA
encoding a secreted protein. Suitable secreted proteins include
hormones, cytokines, growth factors, clotting factors,
anti-protease proteins, angiogenic proteins, antiangiogenic
proteins, chemokines, and antibodies. In particular variations, the
secreted protein is selected from erythropoietin (EPO),
thrombopoietin (TPO), granulocyte-colony stimulating factor
(G-CSF), granulocyte macrophage-colony stimulating factor,
(GM-CSF), leptin, a platelet-derived growth factor (e.g.,
platelet-derived growth factor B (PDGF-B)), keratinocyte growth
factor (KGF), bone morphogenic protein 2 (BMP-2), bone morphogenic
protein 7 (BMP-7), insulin, glucagon-like peptide-1 (GLP-1), human
growth hormone (HGF), Factor VII, Factor VIII, Factor IX, a relaxin
(e.g., relaxin-2), an interferon (e.g., interferon-.alpha.
(IFN-.alpha.), interferon-f (IFN-f), interferon-.gamma.
(IFN-.gamma.)), interleukin-2 (IL-2), interleukin-4 (IL-4),
interleukin-10 (IL-10), interleukin-11 (IL-11), interleukin-12
(IL-12), interleukin-18 (IL-18), interleukin-21 (IL-21), a CC
subfamily chemokine, a CXC subfamily chemokine, a C subfamily
chemokine, and a CX3C subfamily chemokine. In some embodiments
where the secreted protein is an antibody, the antibody is a
genetically engineered antibody selected from a chimeric antibody,
a humanized antibody, a single-chain antibody (e.g., a single-chain
Fv (scFv)), and a bispecific antibody.
[0087] In other embodiments where the therapeutic agent is a
polynucleotide, the polynucleotide is an oligonucleotide. Suitable
oligonucleotide therapeutic agents include siRNAs, antisense
oligonucleotides, anti-miRs (also known as antagomiRs), locked
nucleic acid (LNA)-based oligonucleotides, dicer substrates,
miRNAs, aiRNAs, shRNAs, ribozymes, and nucleic acid aptamers.
[0088] In certain embodiments, the therapeutic agent is a protein,
such as, e.g., an antibody or a peptide aptamer. Particular
variations of antibody therapeutic agents include single chain
antibodies and a bispecific antibodies.
[0089] In some embodiments, the therapeutic agent is a peptide.
Exemplary peptide therapeutic agents include peptide vaccines
comprising one or more short or long amino acid sequences from
disease-associated antigens (e.g., tumor antigens).
[0090] In other embodiments, the therapeutic agent is a small
molecule. In specific variations, the small molecule is selected
from an anti-tubulin agent, a DNA minor groove binding agent, and a
DNA replication inhibitor. In other variations, the small molecule
is selected from an anthracycline, an auristatin, a camptothecin, a
duocarmycin, an etoposide, a maytansinoid, a vinca alkaloid, and a
platinum (II) compound.
[0091] In other embodiments, the therapeutic agent is a component
of a gene editing system that disrupts or corrects a gene
associated with a disease. In some embodiments, the component of
the gene editing system is a polynucleotide (e.g., an mRNA)
encoding a nuclease. Particularly suitable nucleases include zinc
finger nucleases (ZFNs), transcription activator-like effector
nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and
engineered meganucleases. In particular variations in which the
nuclease is Cas9, the lipid nanoparticle further includes a guide
RNA that targets the nuclease to a specific site in the target cell
genome. In some variations directed to gene editing as above, the
lipid nanoparticle further includes a polynucleotide containing a
DNA donor sequence for correcting a disease-associated gene by
homologous recombination. In other variations, the method further
includes administering to the subject an effective amount of a
second lipid nanoparticle that includes a polynucleotide containing
a DNA donor sequence for correcting a disease-associated gene by
homologous recombination.
[0092] In some embodiments, the therapeutic agent is an immunogen.
Suitable immunogens include peptides, proteins, mRNAs, short RNAs,
DNAs, and simple or complex carbohydrates. In certain variations,
the immunogen is derived from an infectious agent (e.g., a virus or
bacteria) or a cancer cell. In some such embodiments, the membrane
destabilizing polymer is also associated with an immunogen, which
may be the same or different than the immunogen of the lipid
nanoparticle.
[0093] In certain embodiments of a method as above where the
therapeutic agent is a polynucleotide, the lipid nanoparticle
includes a mixture of lipid components comprising (i) a cationic
lipid that is permanently charged at physiological pH, where the
cationic lipid is present in the mixture from about 35 mole % to
about 55 mole %; (ii) an ionizable anionic lipid, where the anionic
lipid is optionally absent and, if present, is present in the
mixture from about 25 mole % to about 40 mole %; (iii) a helper
lipid, where if the ionizable anionic lipid is absent, then the
helper lipid is present in the mixture from about 40 mole % to
about 50 mole %, and if the ionizable anionic lipid is present,
then the helper lipid is present in the mixture from about 5 mole %
to about 20 mole %; and (iv) a PEG-lipid, where the PEG-lipid is
present in the mixture from about 2 mole % to about 15 mole %. In
some such embodiments, the cationic lipid is DOTAP, the ionizable
anionic lipid is CHEMS, the helper lipid is CHOL, and/or the
PEG-lipid is DSPE-PEG2k or DMPE-PEG2k. In some variations of a
method comprising a lipid nanoparticle as above, the ionizable
anionic lipid is absent, the cationic lipid is present from about
35 mole % to about 45 mole %, and the PEG-lipid is present from
about 5% mole % to about 15 mole %. In other variations, the
ionizable anionic lipid is present, and the cationic lipid is
present from about 40 mole % to about 55 mole %; in some such
variations, the PEG-lipid is present from about 5 mole % to about
15 mole %. In more specific embodiments, (a) the cationic lipid is
DOTAP, the ionizable anionic lipid is absent, the helper lipid is
CHOL, the PEG-lipid is DSPE-PEG2k, and the molar ratio of
DOTAP:CHOL:DSPE-PEG2k is about 40:50:10; (b) the cationic lipid is
DOTAP, the ionizable anionic lipid is CHEMS, the helper lipid is
CHOL, the PEG-lipid is DMPE-PEG2k, and the molar ratio of
DOTAP:CHEMS:CHOL:DMPE-PEG2k is about 50:32:16:2; (c) the cationic
lipid is DOTAP, the ionizable anionic lipid is CHEMS, the helper
lipid is CHOL, the PEG-lipid is DSPE-PEG2k, and the molar ratio of
DOTAP:CHEMS:CHOL:DSPE-PEG2k is about 50:32:8:10; or (d) the
cationic lipid is DOTAP, the ionizable anionic lipid is CHEMS, the
helper lipid is CHOL, the PEG-lipid is DMPE-PEG2k, and the molar
ratio of DOTAP:CHEMS:CHOL:DMPE-PEG2k is about 50:32:8:10.
[0094] In another aspect, the present invention provides a
composition for delivering a therapeutic or diagnostic agent to the
cytosol of a target cell within a subject. The composition
generally includes (a) a lipid nanoparticle comprising the
therapeutic or diagnostic agent and (b) a membrane-destabilizing
polymer. In some embodiments, at least one of the lipid
nanoparticle and membrane-destabilizing polymer includes a first
targeting ligand that specifically binds to a molecule on the
surface of the target cell. The lipid nanoparticle,
membrane-destabilizing polymer, therapeutic agent, and/or targeting
ligand(s) of the composition include the various embodiments
described above with respect to a method for delivering a
therapeutic or diagnostic agent to a cell.
[0095] In yet another aspect, the present invention provides a
delivery system for delivering a therapeutic or diagnostic agent to
the cytosol of a target cell within a subject. The system generally
includes (a) a carrier composition comprising a lipid nanoparticle,
wherein the lipid nanoparticle comprises the therapeutic or
diagnostic agent, and (b) an enhancer composition comprising a
membrane-destabilizing polymer. In some embodiments, at least one
of the lipid nanoparticle and membrane-destabilizing polymer
includes a first targeting ligand that specifically binds to a
molecule on the surface of the target cell. The lipid nanoparticle,
membrane-destabilizing polymer, therapeutic agent, and/or targeting
ligand(s) of the composition include the various embodiments
described above with respect to a method for delivering a
therapeutic or diagnostic agent to a cell.
[0096] In still another aspect, the present invention provides a
method for treating a disease characterized by a genetic defect
that results in a deficiency of a functional protein. The method
generally includes administering to a subject having the disease
(a) an effective amount of a lipid nanoparticle comprising an mRNA
that encodes the functional protein or a protein having the same
biological activity as the functional protein and (b) an effective
amount of a membrane-destabilizing polymer, where the mRNA is
delivered to the cytosol of target cells of a target tissue
associated with the disease, and where the mRNA is translated
during protein synthesis so as to produce the encoded protein
within the target tissue, thereby treating the disease. In some
embodiments, at least one of the lipid nanoparticle and
membrane-destabilizing polymer comprises a first targeting ligand
that specifically binds to a molecule on the surface of the target
cells of the target tissue. The lipid nanoparticle and
membrane-destabilizing polymer can be administered separately
(e.g., the membrane-destabilizing polymer administered after
administration of the lipid nanoparticle) or, alternatively,
together within a single composition. The lipid nanoparticle and
membrane-destabilizing polymer include the various embodiments
described above with respect to a method for delivering a
therapeutic or diagnostic agent to a cell, provided that the
therapeutic agent is the mRNA, the lipid nanoparticle includes a
cationic lipid (e.g., an ionizable cationic lipid), and the
targeting ligand, if present, is selected to bind to the target
cells of the target tissue exhibiting the protein deficiency. In
certain variations, the lipid nanoparticle and the
membrane-destabilizing polymer are administered in a repeat dosage
regime (e.g., a weekly or bi-weekly repeated administration
protocol).
[0097] In certain embodiments, the disease is a protein deficiency
disease of the liver. In some such embodiments, the mRNA encodes a
functional protein selected from alpha-1-antitrypsin (A1AT),
carbamoyl phosphate synthetase I (CPS1), fumarylacetoacetase (FAH)
enzyme, alanine:glyoxylate-aminotransferase (AGT), methylmalonyl
CoA mutase (MUT), propionyl CoA carboxylase alpha subunit (PCCA),
propionyl CoA carboxylase beta subunit (PCCB), a subunit of
branched-chain ketoacid dehydrogenase (BCKDH), ornithine
transcarbamylase (OTC), copper-transporting ATPase Atp7B, bilirubin
uridinediphosphate glucuronyltransferase (BGT) enzyme, hepcidin,
glucose-6-phosphatase (G6Pase), glucose 6-phosphate translocase,
lysosomal glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1),
Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor
IX, galactose-1-phosphate uridylyltransferase, galactokinase,
UDP-galactose 4-epimerase, transthyretin, a complement regulatory
protein, phenylalanine hydroxylase (PAH), homogentisate
1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine
phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL),
argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1,
alpha-galactosidase A, acid ceramidase, acid .alpha.-L-fucosidase,
acid .beta.-galactosidase, iduronate-2-sulfatase,
alpha-L-iduronidase, galactocerebrosidase, acid
.alpha.-mannosidase, .beta.-mannosidase, arylsulfatase B,
arylsulfatase A, N-acetylgalactosamine-6-sulfate sulfatase, acid
.beta.-galactosidase, acid .alpha.-glucosidase,
.beta.-hexosaminidase B, heparan-N-sulfatase,
alpha-N-acetylglucosaminidase, acetyl-CoA:.alpha.-glucosaminide
N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase,
alpha-N-acetylgalactosaminidase, sialidase, .beta.-glucuronidase,
and .beta.-hexosaminidase A.
[0098] In other embodiments in which the disease is a protein
deficiency disease of the liver, the disease is a urea cycle
disorder. In some such embodiments, the urea cycle disorder is
selected from ornithine transcarbamylase (OTC) deficiency,
carbamoyl phosphate synthetase I (CPS1) deficiency,
argininosuccinic aciduria (argininosuccinate lyase (ASL)
deficiency), and citrullinemia (argininosuccinate synthetase (ASS1)
deficiency). In certain variations where the urea cycle disorder is
ornithine transcarbamylase (OTC) deficiency, the mRNA encodes a
functional OTC protein comprising an amino acid sequence having at
least 90% or at least 95% sequence identity with residues 35-354 of
SEQ ID NO:1. In certain variations where the urea cycle disorder is
argininosuccinic aciduria (argininosuccinate lyase (ASL)
deficiency), the mRNA encodes a functional ASL protein comprising
an amino acid sequence having at least 90% or at least 95% sequence
identity with SEQ ID NO:48. In certain variations where the urea
cycle disorder is citrullinemia (argininosuccinate synthetase
(ASS1) deficiency), the mRNA encodes a functional ASS1 protein
comprising an amino acid sequence having at least 90% or at least
95% sequence identity with SEQ ID NO:50.
[0099] In certain embodiments for treating a protein deficiency
disease of the liver as above, at least one of the
membrane-destabilizing polymer and the lipid nanoparticle comprises
a targeting ligand that specifically binds to the
asialoglycoprotein receptor (ASGPR). Particularly suitable
ASGPR-specific targeting ligands comprise an N-acetylgalactosamine
(NAG) residue.
[0100] In another aspect, the present invention provides a
pH-sensitive, membrane-destabilizing polymer. In some embodiments,
the pH-sensitive, membrane-destabilizing polymer comprises a random
block copolymer of formula Ia:
##STR00002## [0101] wherein [0102] A.sub.0, A.sub.1, A.sub.2,
A.sub.3, A.sub.4 and A.sub.5 are each independently selected from
the group consisting of --C--C--, --C(O)(C).sub.aC(O)O--,
--O(C).sub.aC(O)--, --O(C).sub.b--, and --CR.sub.8--CR.sub.9--;
where tetravalent carbon atoms of A.sub.0-A.sub.5 that are not
fully substituted with R.sub.1-R.sub.6 and Y.sub.0-Y.sub.5 are
completed with an appropriate number of hydrogen atoms; wherein a
and b are each independently 1-4; and where R.sub.8 and R.sub.9 are
each independently selected from the group consisting of --C(O)OH,
--C(O)Oalkyl, and --C(O)NR.sub.10, where R.sub.8 and R.sub.9 are
optionally covalently linked together to form a ring structure;
[0103] Y.sub.5 is hydrogen or is selected from the group consisting
of -(1C-10C)alkyl, -(3C-6C)cycloalkyl, --O-(1C-10C)alkyl,
--C(O)O(1C-10C)alkyl, --C(O)NR.sub.11(1C-10C)alkyl, and
-(6C-10C)aryl, any of which is optionally substituted with one or
more fluorine atoms; [0104] Y.sub.0, Y.sub.3, and Y.sub.4 are each
independently selected from the group consisting of a covalent
bond, -(1C-10C)alkyl-, --C(O)O(2C-10C)alkyl-,
--OC(O)(1C-10C)alkyl-, --O(2C-10C)alkyl-, --S(2C-10C)alkyl-, and
--C(O)NR.sub.12(2C-10C) alkyl-; [0105] Y.sub.1 and Y.sub.2 are each
independently selected from the group consisting of a covalent
bond, -(1C-18C)alkyl-, -(3C-18C)branched alkyl,
--C(O)O(2C-18C)alkyl-, --C(O)O(2C-18C)branched alkyl,
--OC(O)(1C-18C)alkyl-, --OC(O)(1C-18C)branched alkyl-,
--O(2C-18C)alkyl-, --O(2C-18C)branched alkyl-, --S(2C-18C)alkyl-,
--S(2C-18C)branched alkyl-, --C(O)NR.sub.12(2C-18C)alkyl-, and
--C(O)NR.sub.12(2C-18C)branched alkyl-, where any alkyl or branched
alkyl group of Y.sub.1 or Y.sub.2 is optionally substituted with
one or more fluorine atoms;
[0106] R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6,
R.sub.8, R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each
independently hydrogen, --CN, or selected from the group consisting
of alkyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl
and heteroaryl, any of which is optionally substituted with one or
more fluorine atoms; [0107] Q.sub.0 is a residue selected from the
group consisting of residues which are hydrophilic at physiologic
pH; O--[(C).sub.2-3--O].sub.x--R.sub.7; and
O--[(C).sub.2-3--O].sub.x--C(O)--NR.sub.13R.sub.14; where x is
1-48; R.sub.7 is --CH.sub.3 or --CO.sub.2H; and R.sub.13 and
R.sub.14 are each independently hydrogen, --CN, or selected from
the group consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; [0108] Q.sub.1 and
Q.sub.2 are each independently absent or selected from a residue
which is hydrophilic at normal physiological pH; a conjugatable or
functionalizable residue; a residue which is hydrophobic at normal
physiological pH; an alkyl group optionally substituted with one or
more fluorine atoms; and a branched alkyl group optionally
substituted with one or more fluorine atoms; [0109] Q.sub.3 is a
residue which is positively charged at normal physiological pH;
[0110] Q.sub.4 is a residue which is negatively charged at normal
physiological pH, but undergoes protonation at lower pH; [0111] m
is a mole fraction of greater than 0.5 to less than 1.0; [0112] n
is a mole fraction of greater than 0 to less than 0.5; [0113] p is
a mole fraction of 0 to less than 0.5; wherein m+n+p=1; [0114] q is
a mole fraction of 0.1 to 0.9; [0115] r is a mole fraction of 0.05
to 0.9; [0116] s is present up to a mole fraction of 0.85; wherein
q+r+s=1; [0117] v is from 1 to 25 kDa; [0118] w is from 1 to 50
kDa; and
[0119] at least one of Y.sub.1 and Q.sub.1 contains the alkyl or
branched alkyl group substituted with the one or more fluorine
atoms.
[0120] In some embodiments of a pH-sensitive polymer comprising a
copolymer of formula Ia as above, p is 0.
[0121] In some embodiments of a pH-sensitive polymer comprising a
copolymer of formula Ia as above, R.sub.2-A.sub.1-Y.sub.1-Q.sub.1
taken together is a methacrylate residue selected from the group
consisting of 2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue;
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl
2-methylacrylate residue; 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue (also referred to as 2-propenoic acid,
2-methyl-, 3,3,4,4,5,5,6,6,6-nonafluorohexyl ester residue);
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue; and
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue.
[0122] In some embodiments of a pH-sensitive polymer comprising a
copolymer of formula Ia as above, [0123] (a) Y.sub.3 is
--C(O)OCH.sub.2CH.sub.2, Q.sub.3 is dimethylamino, and/or R.sub.4
is --CH.sub.3; [0124] (b) Y.sub.4 is a covalent bond, Q.sub.4 is a
carboxyl residue, and/or R.sub.5 is --CH.sub.2CH.sub.2CH.sub.3;
[0125] (c) Y.sub.5 is --C(O)O(CH.sub.2).sub.3CH.sub.3 and/or
R.sub.6 is --CH.sub.3; and/or [0126] (d) Y.sub.0 is
--C(O)O(2C-10C)alkyl-, Q.sub.0 is
O--[(C).sub.2-3--O].sub.x--R.sub.7 (where x is 1-48 and R.sub.7 is
--CH.sub.3), and/or R.sub.1 is --CH.sub.3.
[0127] In certain embodiments of a pH-sensitive polymer comprising
a copolymer of formula Ia as above, the pH-sensitive polymer is a
polymer of formula Va:
T1-L-[PEGMA.sub.m-M2.sub.n].sub.v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub-
.w Va [0128] where [0129] PEGMA is polyethyleneglycol methacrylate
residue with 2-20 ethylene glycol units; [0130] M2 is a
methacrylate residue selected from the group consisting of [0131] a
(C4-C18)alkyl-methacrylate residue substituted with one or more
fluorine atoms, and [0132] a (C4-C18)branched alkyl-methacrylate
residue substituted with one or more fluorine atoms, [0133] BMA is
butyl methacrylate residue; [0134] PAA is propyl acrylic acid
residue; [0135] DMAEMA is dimethylaminoethyl methacrylate residue;
[0136] m and n are each a mole fraction greater than 0, where m is
greater than n and m+n=1; [0137] q is a mole fraction of 0.2 to
0.75; [0138] r is a mole fraction of 0.05 to 0.6; [0139] s is a
mole fraction of 0.2 to 0.75; [0140] q+r+s=1; [0141] v is 1 to 25
kDa; [0142] w is 1 to 25 kDa; [0143] T1 is absent or is the first
targeting ligand; and [0144] L is absent or is a linking
moiety.
[0145] In certain variations of a pH-sensitive polymer of formula
Va as above, M2 is a methacrylate residue selected from the group
consisting of 2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue;
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl
2-methylacrylate residue; 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue; and
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue; and
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue.
[0146] In other embodiments, a pH-sensitive, membrane-destabilizing
polymer is a polymer of formula V:
T1-L-[PEGMA.sub.m-M2n].sub.v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub.w
V [0147] where [0148] PEGMA is polyethyleneglycol methacrylate
residue with 2-20 ethylene glycol units; [0149] M2 is a
methacrylate residue selected from the group consisting of [0150] a
(C4-C18)alkyl-methacrylate residue; [0151] a (C4-C18)branched
alkyl-methacrylate residue; [0152] a cholesteryl methacrylate
residue; [0153] a (C4-C18)alkyl-methacrylate residue substituted
with one or more fluorine atoms; and [0154] a (C4-C18)branched
alkyl-methacrylate residue substituted with one or more fluorine
atoms; [0155] BMA is butyl methacrylate residue; [0156] PAA is
propyl acrylic acid residue; [0157] DMAEMA is dimethylaminoethyl
methacrylate residue; [0158] m and n are each a mole fraction
greater than 0, wherein m is greater than n and m+n=1; [0159] q is
a mole fraction of 0.2 to 0.75; [0160] r is a mole fraction of 0.05
to 0.6; [0161] s is a mole fraction of 0.2 to 0.75; [0162] q+r+s=1;
[0163] v is 1 to 25 kDa; [0164] w is 1 to 25 kDa; [0165] T1 is
absent or is the first targeting ligand; and [0166] L is absent or
is a linking moiety.
[0167] In certain variations of a pH-sensitive polymer of formula V
as above, M2 is a methacrylate residue selected from the group
consisting of 2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue;
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl
2-methylacrylate residue; 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue; 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl
methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue;
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue; 2-ethylhexyl methacrylate residue; butyl
methacrylate residue; hexyl methacrylate residue; octyl
methacrylate residue, n-decyl methacrylate residue; lauryl
methacrylate residue; myristyl methacrylate residue; stearyl
methacrylate residue; cholesteryl methacrylate residue; ethylene
glycol phenyl ether methacrylate residue; 2-propenoic acid,
2-methyl-, 2-phenylethyl ester residue; 2-propenoic acid,
2-methyl-, 2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl ester
residue; 2-propenoic acid, 2-methyl-, 2-(1H-imidazol-1-yl)ethyl
ester residue; 2-propenoic acid, 2-methyl-, cyclohexyl ester
residue; 2-propenoic acid, 2-methyl-,
2-[bis(1-methylethyl)amino]ethyl ester residue; 2-propenoic acid,
2-methyl-, 3-methylbutyl ester residue; neopentyl methacrylate
residue; tert-butyl methacrylate residue; 3,3,5-trimethyl
cyclohexyl methacrylate residue; 2-hydroxypropyl methacrylate
residue; 5-nonyl methacrylate residue; 2-butyl-1-octyl methacrylate
residue; 2-hexyl-1-decyl methacrylate residue; and 2-(tert-butyl
amino)ethyl methacrylate residue.
[0168] In yet another aspect, the present invention provides a
lipid nanoparticle. In some embodiments, the lipid nanoparticle
comprises (a) a polynucleotide, and (b) a mixture of lipid
components comprising (i) a cationic lipid that is permanently
charged at physiological pH, where the cationic lipid is present in
the mixture from about 35 mole % to about 55 mole %; (ii) an
ionizable anionic lipid, where the anionic lipid is optionally
absent and, if present, is present in the mixture from about 25
mole % to about 40 mole %; (iii) a helper lipid, where if the
ionizable anionic lipid is absent, then the helper lipid is present
in the mixture from about 40 mole % to about 50 mole %, and if the
ionizable anionic lipid is present, then the helper lipid is
present in the mixture from about 5 mole % to about 20 mole %; and
(iv) a PEG-lipid, where the PEG-lipid is present in the mixture
from about 5 mole % to about 15 mole %. In some such embodiments,
the cationic lipid is DOTAP, the ionizable anionic lipid is CHEMS,
the helper lipid is CHOL, and/or the PEG-lipid is DSPE-PEG2k or
DMPE-PEG2k. In some variations of a lipid nanoparticle as above,
the ionizable anionic lipid is absent and the cationic lipid is
present from about 35 mole % to about 45 mole %. In other
variations, the ionizable anionic lipid is present, and the
cationic lipid is present from about 40 mole % to about 55 mole %.
In more specific embodiments, (a) the cationic lipid is DOTAP, the
ionizable anionic lipid is absent, the helper lipid is CHOL, the
PEG-lipid is DSPE-PEG2k, and the molar ratio of
DOTAP:CHOL:DSPE-PEG2k is about 40:50:10; (b) the cationic lipid is
DOTAP, the ionizable anionic lipid is CHEMS, the helper lipid is
CHOL, the PEG-lipid is DSPE-PEG2k, and the molar ratio of
DOTAP:CHEMS:CHOL:DSPE-PEG2k is about 50:32:8:10; or (c) the
cationic lipid is DOTAP, the ionizable anionic lipid is CHEMS, the
helper lipid is CHOL, the PEG-lipid is DMPE-PEG2k, and the molar
ratio of DOTAP:CHEMS:CHOL:DMPE-PEG2k is about 50:32:8:10. In
certain embodiments of a lipid nanoparticle as above, the
polynucleotide is an mRNA.
[0169] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention.
Definitions
[0170] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art pertinent to the methods and compositions
described. As used herein, the following terms and phrases have the
meanings ascribed to them unless specified otherwise.
[0171] The terms "a," "an," and "the" include plural referents,
unless the context clearly indicates otherwise.
[0172] As used herein, the term "lipid nanoparticle" or "LNP"
refers to a particle of less than about 1,000 nm, typically less
than about 200 nm, that is formulated with at least one lipid
molecular species. Lipid nanoparticles include (but are not limited
to) liposomes, irrespective of their lamellarity, shape, or
structure. As used herein, a "liposome" is a structure having
lipid-containing membranes enclosing an aqueous interior. Liposomes
may have one or more lipid membranes. Single-layered liposomes are
referred to as "unilamellar," and multi-layered liposomes are
referred to as "multilamellar." Lipid nanoparticles may further
include one or more additional lipids and/or other components,
which may be included in the liposome compositions for a variety of
purposes, such as to stabilize a lipid membrane, to prevent lipid
oxidation, or to attach ligands on the liposome surface. Any number
of lipids may be present, including amphipathic, neutral, cationic,
and anionic lipids. Lipid nanoparticles can be complexed with
therapeutic or diagnostic agents, including polynucleotides,
proteins, peptides, or small molecules, and are useful as in vivo
delivery vehicles.
[0173] The term "cationic lipid" refers to any of a number of lipid
species which carry a net positive charge at physiological pH. Such
lipids include, but are not limited to, DODAC, DOTMA, DOTAP,
DC-Chol, DMRIE, DOEPC, DLEPC, DMEPC, 14:1, MVL5, DOGS, DORIE, DORI,
and DILA.sup.2.
[0174] The term "neutral lipid" refers to any of a number of lipid
species that exist either in an uncharged or neutral zwitterionic
form at physiological pH. Such lipids include, for example
cholesterol, DOPE, DLPE, DLPC, phosphatidylcholines,
phosphatidylethanolamines, phosphatidylserines, ceramide,
sphingomyelin, cephalin, and cerebrosides.
[0175] The term "non-cationic lipid" refers to any neutral lipid as
described above as well as anionic lipids (i.e., lipid species that
carry a net negative charge at physiological pH). Examples of
anionic lipids include, but are not limited to, cardiolipin,
phosphatidylserine and phosphatidic acid.
[0176] An "ionizable anionic lipid" means an anionic lipid that
undergoes protonation as the pH is reduced toward the pK.sub.a of
the lipid. At the pK.sub.a of the ionizable anionic lipid, half of
the lipid is in the anionic form and half of the lipid is in the
protonated form. In the context of lipid nanoparticles, at pH
values above the pK.sub.a of the ionizable anionic lipid, more of
the lipid is negatively charged, and the negatively charged form of
the lipid can stabilize other lipids in a bilayer organization,
allowing the formation of bilayer vesicles. These vesicles then
fuse as the pH is reduced toward the pK.sub.a of the ionizable
anionic lipid, such as in the endosomal environment, and more of
the ionizable anionic lipid becomes protonated. Examples of
ionizable anionic lipids include cholesteryl hemisuccinate (CHEMS),
phosphatidylserine, palmitoylhomoserine, and .alpha.-tocopherol
hemisuccinate.
[0177] An "ionizable cationic lipid" means a cationic lipid that
undergoes protonation as the pH is reduced toward the pK.sub.a of
the lipid. At the pK.sub.a of the ionizable cationic lipid, half of
the lipid in in the protonated form and half of the lipid is in the
neutral form. In the context of lipid nanoparticles, at pH values
below the pK.sub.a of the ionizable cationic lipid, the positively
charged form of the lipid can interact with negatively charged
oligonucleotides, allowing for encapsulation of the
oligonucleotides inside of vesicles and nanoparticles. At pH values
above the pK.sub.a, more of the cationic lipid is neutral and this
lack of charge can affect the surface potential of lipid
nanoparticles as well as affect release of oligonucleotides from
these lipids. Additionally, appropriately designed cationic lipids
with unsaturated tails can mediate fusion events with other
membranes by undergoing lamellar to inverse hexagonal phase
transitions. Such fusion events can result in endosomolysis which
can enable delivery of material into the cytosol. Examples of
ionizable anionic lipids include DDAB, DlinDMA, DLin-KC2-DMA, MC3
lipid (DLin-MC3-DMA), DODAP, DODMA, and Mo-CHOL.
[0178] An "exchangeable PEG-lipid" means a PEG-lipid that is not
stable in a lipid nanoparticle (LNP) membrane at physiologic
temperature, such that PEG-lipid molecules in the LNP leave the LNP
membrane over time. Exchangeable PEG-lipids leaving the LNP
membrane typically move into a biological membrane (e.g., blood
cell membranes) or may form micelles by themselves. The rate of
release of a PEG-lipid from an LNP is mainly a function of the
length of the alkyl chain and the level of unsaturation in the
alkyl chain (i.e., the number of carbon-to-carbon double bonds).
Typically, a PEG-lipid having a saturated chain of 14 carbons or
less will be exchangeable. A C18 chain with one or more double
bonds (e.g., 18:1, 18:2) will also be exchangeable. Generally, a
PEG-lipid having an alkyl chain of greater than 18 carbons will not
be exchangeable or exchanges at a much lower rate than a PEG-lipid
having an alkyl chain of 14 carbons or less. Other factors that can
increase the rate of release of a PEG-lipid include asymmetry in
the alkyl chain (e.g., PEG-Ceramides with different alkyl chain
lengths (e.g., cerC8)) as well as the size of the PEG moiety, with
larger molecular weight PEG moieties contributing to
exchangeability of the lipid.
[0179] As used herein, "amphipathic" or "amphiphilic" compounds
have both hydrophilic (water-soluble) and hydrophobic
(water-insoluble) parts.
[0180] As used herein, the term "therapeutic agent" refers to any
molecular species (e.g., polynucleotide, protein, peptide, or small
molecule) that may have a therapeutic effect upon delivery into a
cell. In the case of a polynucleotide, this effect can be mediated
by the nucleic acid itself (e.g., anti-sense polynucleotide),
following transcription (e.g., anti-sense RNA, ribozymes,
interfering dsRNA, mRNA), or following expression into a protein. A
"therapeutic" effect of an expressed protein in attenuating or
preventing the disease state can be accomplished by the protein
either staying within the cell, remaining attached to the cell in
the membrane, or being secreted and dissociated from the cell where
it can enter the general circulation and blood. Secreted proteins
that can be therapeutic include hormones, cytokines, growth
factors, clotting factors, anti-protease proteins (e.g.,
alpha1-antitrypsin), angiogenic proteins (e.g., vascular
endothelial growth factor, fibroblast growth factors),
antiangiogenic proteins (e.g., endostatin, angiostatin), and other
proteins that are present in the blood. Proteins on the membrane
can have a therapeutic effect by providing a receptor for the cell
to take up a protein or lipoprotein. Therapeutic proteins that stay
within the cell (intracellular proteins) can be enzymes that clear
a circulating toxic metabolite as in phenylketonuria. They can also
cause a cancer cell to be less proliferative or cancerous (e.g.,
less metastatic), or interfere with the replication of a virus.
Intracellular proteins can be part of the cytoskeleton (e.g.,
actin, dystrophin, myosins, sarcoglycans, and dystroglycans) and
thus have a therapeutic effect in cardiomyopathies and
musculoskeletal diseases (e.g., Duchenne muscular dystrophy,
limb-girdle disease). Protein agents may also be delivered directly
into a cell (i.e., in protein form, rather than as an encoding
polynucleotide to be expressed). Other therapeutic proteins of
particular interest to treating heart disease include polypeptides
affecting cardiac contractility (e.g., calcium and sodium
channels), inhibitors of restenosis (e.g., nitric oxide
synthetase), angiogenic factors, and anti-angiogenic factors.
Protein agents may also include antibodies (e.g., small
single-chain antibodies or bispecific antibodies) directed at
intracellular targets. Other exemplary "therapeutic agents" include
small molecules, such as, for example, small molecule inhibitors or
agonists of intracellular target molecules (e.g., kinase
inhibitors, inhibitors of DNA synthesis pathways) or small
molecules having a cytotoxic or cytostatic effect on a cell (such
as chemotherapeutic agents for cancer treatment); anti-infective
agents (e.g., anti-viral agents or anti-bacterial agents); or
vaccines (which may include proteins, peptides, DNA, or RNA). In
some embodiments, a "therapeutic agent" is a component of a gene
editing system that disrupts or corrects genes that cause disease
(e.g., a polynucleotide encoding a nuclease; a guide RNA that may
be formulated with a polynucleotide encoding a nuclease; or a donor
DNA sequence for correcting a gene by homologous
recombination).
[0181] As used herein, the term "diagnostic agent" refers to a
component that can be detected in a subject or test sample from a
subject. Exemplary diagnostic agents include radioactive agents,
fluorescent agents, contrast agents (e.g., an MRI or X-ray contrast
agent), and other imaging reagents. Diagnostic reagents also
include, for example, immunodiagnostic reagents (e.g., antibodies
directed to intracellular targets) as well as other specific
binding agents. A diagnostic agent may consist of, for example, a
diagnostically detectable label that is complexed with a lipid
nanoparticle, or may comprise a diagnostically detectable label
conjugated to another molecule (e.g., a specific binding molecule,
such as, e.g., a peptide, protein, or polynucleotide). Many
different labels exist in the art and methods of labeling are
well-known by the skilled artisan. General classes of labels that
can be used in the present invention include, but are not limited
to, radioactive isotopes, paramagnetic isotopes, compounds that can
be imaged by positron emission tomography (PET), fluorescent or
colored compounds, compounds which can be imaged by magnetic
resonance, chemiluminescent compounds, bioluminescent compounds,
and the like. Particularly suitable detectable labels include, but
are not limited to, radioactive, fluorescent, fluorogenic, or
chromogenic labels. Useful radiolabels (radionuclides), which are
detected simply by .gamma. counter, scintillation counter or
autoradiography include, but are not limited to, .sup.3H,
.sup.125I, .sup.131I, .sup.35S, and .sup.14C.
[0182] As used herein, the term "membrane-destabilizing polymer"
refers to a polymer that is capable of inducing one or more of the
following effects upon a biological membrane: an alteration or
disruption that allows small molecule permeability, pore formation
in the membrane, a fusion and/or fission of membranes, an
alteration or disruption that allows large molecule permeability, a
dissolving of the membrane, or causing membrane perturbation that
opens tight junctions and enables paracellular transport. This
alteration can be functionally defined by the compound's activity
in at least one the following assays: red blood cell lysis
(hemolysis), liposome leakage, liposome fusion, cell fusion, cell
lysis, and release of endosomal contents. Typically, a
membrane-destabilizing polymer allows for the transport of
molecules with a molecular weight greater than 50 atomic mass units
to cross a membrane. This transport may be accomplished by either
the loss of membrane structure or the formation of holes or pores
in the membrane. In particular variations, a membrane-destabilizing
polymer is a copolymer (e.g., an amphipathic copolymer), a
synthetic amphipathic peptide, a membrane active toxin (e.g.,
pardaxin, melittin, cecropin, magainin, PGLa, indolicidin,
dermaseptin, or a derivative thereof), or a viral fusogenic peptide
(e.g., the influenza virus hemagglutinin subunit HA-2 peptide).
[0183] As used herein, a "block copolymer" refers to a structure
comprising one or more sub-combination of constitutional or
monomeric units. In some embodiments, the block copolymer is a
diblock copolymer, a tri-block copolymer or a higher-ordered block
copolymer. For example, a diblock copolymer can comprise two
blocks; a schematic generalization of such a polymer is represented
by the following: [A.sub.a-B.sub.b-C.sub.c- . . .
].sub.m-[X.sub.x-Y.sub.y-Z.sub.z- . . . ], or
[A.sub.a-B.sub.b-C.sub.c- . . . ].sub.m-b-[X.sub.x-Y.sub.y-Z.sub.z-
. . . ].sub.n, wherein each letter stands for a constitutional or
monomeric unit, and wherein each subscript to a constitutional unit
represents the mole fraction of that unit in the particular block,
the three dots indicate that there may be more (there may also be
fewer) constitutional units in each block, and m and n indicate the
molecular weight (or weight fraction) of each block in the diblock
copolymer. As suggested by such schematic representation, in some
instances, the number and the nature of each constitutional unit is
separately controlled for each block. The schematic is not meant
to, and should not be construed to, infer any relationship
whatsoever between the number of constitutional units or between
the number of different types of constitutional units in each of
the blocks. Nor is the schematic meant to describe any particular
number or arrangement of the constitutional units within a
particular block. In each block the constitutional units may be
disposed in a purely random, an alternating random, a regular
alternating, a regular block or a random block configuration unless
expressly stated to be otherwise. A purely random configuration,
for example, may have the form: x-x-y-z-x-y-y-z-y-z-z-z . . . . An
exemplary alternating random configuration may have the form:
x-y-x-z-y-x-y-z-y-x-z . . . , and an exemplary regular alternating
configuration may have the form: x-y-z-x-y-z-x-y-z . . . . An
exemplary regular block configuration may have the following
general configuration: . . . x-x-x-y-y-y-z-z-z-x-x-x . . . , while
an exemplary random block configuration may have the general
configuration: . . . x-x-x-z-z-x-x-y-y-y-y-z-z-z-x-x-z-z-z- . . . .
In a gradient polymer, the content of one or more monomeric units
increases or decreases in a gradient manner from the .alpha. end of
the polymer to the .omega. end. In none of the preceding generic
examples is the particular juxtaposition of individual
constitutional units or blocks or the number of constitutional
units in a block or the number of blocks meant nor should they be
construed as in any manner bearing on or limiting the actual
structure of block copolymers forming the polymeric carrier of this
invention.
[0184] As used herein, the brackets enclosing the constitutional
units are not meant and are not to be construed to mean that the
constitutional units themselves form blocks. That is, the
constitutional units within the square brackets may combine in any
manner with the other constitutional units within the block, i.e.,
purely random, alternating random, regular alternating, regular
block or random block configurations. The block copolymers
described herein are, optionally, alternate, gradient or random
block copolymers.
[0185] As used herein, the term "molecular weight" for a polymer or
polymer block is the number average molecular weight. It is
understood in the art that a population of polymer molecules will
have a distribution of different molecular weights. This
distribution of molecular weights can be described by the term
dispersity index or polydispersity index (PI or PDI), which is the
weight average molecular weight/number average molecular
weight.
[0186] As used herein the term "polynucleotide" refers to a polymer
comprising two or more nucleotide monomeric units ("nucleotides").
Typical polynucleotides in accordance with certain embodiments of
the present invention include those comprising 7-20,000 nucleotide
monomeric units, 7-15,000 nucleotide monomeric units, 7-10,000
nucleotide monomeric units, 7-5,000 nucleotide monomeric units and
7-1000 nucleotide monomeric units. Polynucleotides of less than 200
nucleotides are generally referred to as "oligonucleotides."
Polynucleotides include deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA), or their derivatives, and combinations of DNA, RNA. DNA
may be in form of cDNA, in vitro polymerized DNA, plasmid DNA,
parts of a plasmid DNA, genetic material derived from a virus,
linear DNA, vectors (Pl, PAC, BAC, YAC, and artificial
chromosomes), expression vectors, expression cassettes, chimeric
sequences, recombinant DNA, chromosomal DNA, anti-sense DNA, or
derivatives of these groups. RNA may be in the form of messenger
RNA (mRNA), in vitro polymerized RNA, recombinant RNA, transfer RNA
(tRNA), small nuclear RNA (snRNA), ribosomal RNA (rRNA), chimeric
sequences, dicer substrate and the precursors thereof, locked
nucleic acids, anti-sense RNA, interfering RNA (RNAi), asymmetric
interfering RNA (aiRNA), small interfering RNA (siRNA), microRNA
(miRNA), ribozymes, external guide sequences, small non-messenger
RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs (24-mers, modified
snmRNA that act by an anti-sense mechanism), tiny non-coding RNAs
(tncRNAs), small hairpin RNA (shRNA), or their derivatives. In
addition, DNA and RNA may be single, double, triple, or quadruple
stranded. Double stranded RNA (dsRNA) and siRNA are of interest
particularly in connection with the phenomenon of RNA interference.
Examples of oligonucleotides as used herein include, but are not
limited to, siRNA, an antisense oligonucleotide, a dicer substrate,
a miRNA, an aiRNA or an shRNA. Further examples of oligonucleotides
as used herein include, but are not limited to dsRNA having a
length of from 17 to 29 nucleotides, or from 19 to 25 nucleotides,
and being at least 90 percent, or 95 percent or 100 percent (of the
nucleotides of a dsRNA) complementary to a coding or a non-coding
section of the nucleic acid sequence of a therapeutically relevant
protein or antigen. Ninety percent complementary means that a 20
nucleotide length of a dsRNA contains not more than 2 nucleotides
without a corresponding complementarity with the corresponding
section of the mRNA. Yet further examples of polynucleotides as
used herein include, but are not limited to single stranded mRNA
which can be modified or unmodified. Modified mRNA includes at
least one modification and a translatable region. Modification(s)
may be located on the backbone, a nucleoside of the nucleic acid
molecule, and/or a 5' cap structure. For example, a modification
may be located on a nucleoside (e.g., substitution of uridine
residues with pseudouridine), or modifications may be located on
both a nucleoside and a backbone linkage. Typically, mRNAs in
accordance with certain compositions and methods of the present
invention include those comprising 300-20,000 nucleotide monomeric
units, 300-15,000 nucleotide monomeric units, 300-10,000 nucleotide
monomeric units, 300-5,000 nucleotide monomeric units, 300-2000
nucleotide monomeric units, 300-1,500 nucleotide monomeric units,
and 300-1000 nucleotide monomeric units. In some variations, an
mRNA in accordance with compositions and methods of the present
disclosure is at least 500, at least 1,000, at least 1,200, or at
least 1,500 nucleotide monomeric units.
[0187] Polynucleotides may include nucleotides that have been
modified relative to naturally occurring nucleotides. Modified
nucleotides can have alterations in sugar moieties and/or in
pyrimidine or purine base moieties. Sugar modifications include,
for example, replacement of one or more hydroxyl groups with
halogens, alkyl groups, amines, and azido groups, or sugars can be
functionalized as ethers or esters. Moreover, the entire sugar
moiety can be replaced with sterically and electronically similar
structures, such as aza-sugars and carbocyclic sugar analogs.
Examples of modifications in a base moiety include alkylated
purines and pyrimidines, acylated purines or pyrimidines, or other
well-known heterocyclic substitutes. Nucleotide monomeric units can
be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "polynucleotide" also includes so-called "peptide
nucleic acids," which comprise naturally-occurring or modified
nucleic acid bases attached to a polyamide backbone.
[0188] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 50 amino acid residues are commonly
referred to as "peptides."
[0189] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0190] With regard to proteins as described herein, reference to
amino acid residues corresponding to those specified by SEQ ID NO
includes post-translational modifications of such residues.
[0191] As used herein, the term "antibody" refers to any
immunoglobulin protein that specifically binds to an antigen, as
well as antigen-binding fragments thereof and engineered variants
thereof. Hence, the term "antibody" includes, for example,
polyclonal antibodies, monoclonal antibodies, and antigen-binding
antibody fragments that contain the paratope of an intact antibody,
such as Fab, Fab', F(ab').sub.2 and F(v) fragments. Genetically
engineered intact antibodies and fragments, such as chimeric
antibodies, humanized antibodies, single-chain Fv fragments,
single-chain antibodies, diabodies, minibodies, linear antibodies,
multivalent or multispecific hybrid antibodies, and the like are
also included. Thus, the term "antibody" is used expansively to
include any protein that comprises an antigen binding site of an
antibody and is capable of binding to its antigen. In some
embodiments, an antibody has affinity to a cell surface
molecule.
[0192] The term "genetically engineered antibodies" means
antibodies wherein the amino acid sequence has been varied from
that of a native antibody. Because of the relevance of recombinant
DNA techniques in the generation of antibodies, one need not be
confined to the sequences of amino acids found in natural
antibodies; antibodies can be redesigned to obtain desired
characteristics. The possible variations are many and range from
the changing of just one or a few amino acids to the complete
redesign of, for example, the variable or constant region. Changes
in the constant region will, in general, be made in order to
improve or alter characteristics, such as complement fixation,
interaction with cells and other effector functions. Typically,
changes in the variable region will be made in order to improve the
antigen binding characteristics, improve variable region stability,
or reduce the risk of immunogenicity.
[0193] An "antigen-binding site of an antibody" is that portion of
an antibody that is sufficient to bind to its antigen. The minimum
such region is typically a variable domain or a genetically
engineered variant thereof. Single-domain binding sites can be
generated from camelid antibodies (see Muyldermans and Lauwereys,
J. Mol. Recog. 12:131-140, 1999; Nguyen et al., EMBO J. 19:921-930,
2000) or from V.sub.H domains of other species to produce
single-domain antibodies ("dAbs"; see Ward et al., Nature
341:544-546, 1989; U.S. Pat. No. 6,248,516 to Winter et al.). In
certain variations, an antigen-binding site is a polypeptide region
having only 2 complementarity determining regions (CDRs) of a
naturally or non-naturally (e.g., mutagenized) occurring heavy
chain variable domain or light chain variable domain, or
combination thereof (see, e.g., Pessi et al., Nature 362:367-369,
1993; Qiu et al., Nature Biotechnol. 25:921-929, 2007). More
commonly, an antigen-binding site of an antibody comprises both a
heavy chain variable domain and a light chain variable domain that
bind to a common epitope. Examples of molecules comprising an
antigen-binding site of an antibody are known in the art and
include, for example, Fv fragments, single-chain Fv fragments
(scFv), Fab fragments, diabodies, minibodies, Fab-scFv fusions,
bispecific (scFv).sub.4-IgG, and bispecific (scFv).sub.2-Fab. (See,
e.g., Hu et al., Cancer Res. 56:3055-3061, 1996; Atwell et al.,
Molecular Immunology 33:1301-1312, 1996; Carter and Merchant, Curr.
Opin. Biotechnol. 8:449-454, 1997; Zuo et al., Protein Engineering
13:361-367, 2000; and Lu et al., J. Immunol. Methods 267:213-226,
2002.)
[0194] As used herein, the terms "single-chain Fv" and
"single-chain antibody" refer to antibody fragments that comprise,
within a single polypeptide chain, the variable regions from both
heavy and light chains, but lack constant regions. In general, a
single-chain antibody further comprises a polypeptide linker
between the V.sub.H and V.sub.L domains, which enables it to form
the desired structure that allows for antigen binding. Single-chain
antibodies are discussed in detail by, for example, Pluckthun in
The Pharmacology of Monoclonal Antibodies, vol. 113 (Rosenburg and
Moore eds., Springer-Verlag, New York, 1994), pp. 269-315. (See
also WIPO Publication WO 88/01649; U.S. Pat. Nos. 4,946,778 and
5,260,203; Bird et al., Science 242:423-426, 1988.) Single-chain
antibodies can also be bi-specific and/or humanized.
[0195] A "bispecific antibody" is a hybrid antibody having two
different heavy/light chain pairs and two different binding sites.
Bispecific antibodies are well-established in the art as a standard
technique to create a single protein that binds to two different
determinants. See, e.g., Kufer et al., Trends Biotechnol.
22:238-244, 2004. Bispecific antibodies may be made in many
different formats, including but not limited to quadroma, F(ab')2,
tetravalent, heterodimeric scFv, bispecific scFv, tandem scFv,
diabody and minibody formats, or scFvs appended to or recombinantly
fused with whole antibodies. See e.g., Kufer et al., 2004; Holliger
and Hudson Nature Biotechnology 23:1126-1136, 2005; Morrison and
Coloma, WO 95/09917.
[0196] As used herein, an "immunogen" is an entity (e.g., a
peptide, protein, a nucleic acid, or a carbohydrate) that induces
an immune response, which may include an innate or an adaptive
immune response (e.g., that protects a subject from an infection or
cancer). An adaptive immune response can be a humoral and/or
cell-mediated immune response. In certain embodiments, an immunogen
in the context of the present disclosure is used as a vaccine.
[0197] As used herein the term "sugar" refers to saccharides such
as monosaccharides, disaccharides, oligosaccharides, and
polysaccharides for example. Typically, sugars as used herein
target or deliver copolymers to target cells or tissues, or
specific cells types and enhance the association of molecules with
the target cells. For example, liver hepatocytes contain
asialoglycoprotein (ASGP) receptors. Therefore,
galactose-containing targeting groups may be used to target
hepatocytes. Examples of galactose containing targeting groups
include, but are not limited to, galactose or galactose derivatives
such as its protected analogs, N-acetylgalactosamine (NAG, also
referred to as GalNAc) or N-acetylgalactosamine derivatives such as
its protected analogs, oligosaccharides, and saccharide clusters
such as Tyr-Glu-Glu-(aminohexyl GalNAc)3, lysine-based galactose
clusters, and cholane-based galactose clusters. Other examples of
sugars include, but are not limited to, mannose and mannose
derivatives such as its protected analogs. In some variations, a
sugar is a multivalent structure comprising two or more sugar
moieties (e.g., three or four moieties). In some such multivalent
sugar embodiments, each moiety is connected to a common branching
point via a linker. An exemplary multivalent sugar is a
tri-N-acetylgalactosamine (tri-NAG) structure having three NAG
moieties. Tri-NAG structures are generally known in the art and are
described, for example, in Lee et al., Carbohydrates and Chemistry
and Biology (B. Ernst, G. W. Hart, & P. Sinay, Eds., Wiley-WCH:
Weinheim, 2000), Vol. 4, p459 (and references cited therein);
Biessen et al. J. Med. Chem. 38:1538, 1995; Sliedregt et al., J.
Med. Chem. 42:609, 1999; Rensen et al., J. Med. Chem. 47:5798,
2004; Khorev et al., Bioorg. Med. Chem. 16:5216, 2008. Another
exemplary multivalent sugar is a bis-mannose-6-phosphate (bis-M6P)
structure having two mannose-6-phosphate moieties (see, e.g., U.S.
Pat. No. 8,399,657 to Zhu et al.).
[0198] As used herein the term "vitamin" refers any of various
fat-soluble or water-soluble organic substances that are essential
in minute amounts for normal growth and activity of living
organisms. Exemplary vitamins include Vitamin A (Retinol), Vitamin
B1 (Thiamine), Vitamin C (Ascorbic acid), Vitamin D (Calciferol),
Vitamin B2 (Riboflavin), Vitamin E (Tocopherol), Vitamin B12
(Cobalamins), Vitamin K1 (Phylloquinone), Vitamin B5 (Pantothenic
acid), Vitamin B7 (Biotin), Vitamin B6 (Pyridoxine), Vitamin B3
(Niacin), Vitamin B9 (Folic acid) and their derivatives. Typically,
vitamins as used herein target or deliver lipid nanoparticles
and/or membrane-destabilizing polymers to target cells or tissues,
or specific cells types and enhance the association of molecules
with the target cells. An example of a vitamin as used herein
includes Vitamin B.sub.9, including folic acid, folate and their
derivatives.
[0199] As used herein, a "targeting ligand" refers to a moiety that
is capable of specifically binding to a molecule on the surface of
a target cell, such as a cell within a target tissue of a subject.
A molecule (e.g., cell surface molecule) that specifically binds to
a targeting moiety is also referred to herein as a "binding
partner."
[0200] As used herein, "alkyl" refers to a straight or branched
chain fully saturated (no double or triple bonds) hydrocarbon
(carbon and hydrogen only) group, optionally having a cycloalkyl
group as part of the hydrocarbon chain (either at a terminal
position or non-terminal position in the chain). An alkyl group
herein contains from one to ten carbon atoms in the principal chain
and up to 20 carbon atoms, and may be linear or branched. Examples
of alkyl groups include, but are not limited to, methyl, ethyl,
propyl, isopropyl, butyl, isobutyl, sec-butyl, tertiary butyl,
pentyl and hexyl. As used herein, "alkyl" includes "alkylene"
groups, which refer to straight or branched fully saturated
hydrocarbon groups having two rather than one open valences for
bonding to other groups. Examples of alkylene groups include, but
are not limited to methylene (--CH.sub.2--), ethylene
(--CH.sub.2CH.sub.2--), propylene (--CH.sub.2CH.sub.2CH.sub.2--),
n-butylene (--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--), sec-butylene
(--CH.sub.2CH.sub.2CH(CH.sub.3)--), and the like. An alkyl group of
this disclosure may optionally be substituted with one or more
fluorine groups.
[0201] As used herein, "mC to nC," "Cm to Cn," or "Cm to C.sub.n,"
wherein m and n are integers, refers to the number of possible
carbon atoms in the indicated group. That is, the group can contain
from "m" to "n", inclusive, carbon atoms. An alkyl group of this
disclosure may comprise from 1 to 18 carbon atoms, that is, m is 1
and n is 18. Of course, a particular alkyl group may be more
limited. For instance without limitation, an alkyl group of this
disclosure may consist of 3 to 8 carbon atoms, in which case it
would be designated as a (3C-8C)alkyl group. The numbers are
inclusive and incorporate all straight or branched chain structures
having the indicated number of carbon atoms. For example without
limitation, a "1C to 4C alkyl" or "(1C-4C)alkyl" group refers to
all alkyl groups having from 1 to 4 carbons, that is, CH.sub.3--,
CH.sub.3CH.sub.2--, CH.sub.3CH.sub.2CH.sub.2--,
CH.sub.3CH(CH.sub.3)--, CH.sub.3CH.sub.2CH.sub.2CH.sub.2--,
CH.sub.3CH.sub.2CH(CH.sub.3)--, (CH.sub.3).sub.2CHCH.sub.2-- and
(CH.sub.3).sub.3CH--.
[0202] As used herein, the term "aryl" or "aryl group" refers to
optionally substituted monocyclic, bicyclic, and tricyclic ring
systems having a total of five to fourteen ring members, wherein at
least one ring in the system is aromatic and wherein each ring in
the system contains three to seven ring members. The terms "aryl"
or "ar" as used herein alone or as part of another group denote
optionally substituted homocyclic aromatic groups, preferably
monocyclic or bicyclic groups containing from 6 to 12 carbons in
the ring portion, such as phenyl, biphenyl, naphthyl, substituted
phenyl, substituted biphenyl or substituted naphthyl. Phenyl and
substituted phenyl are the more preferred aryl.
[0203] As used herein, the term "heteroalkyl" means an alkyl group
wherein at least one of the backbone carbon atoms is replaced with
a heteroatom.
[0204] As used herein, the term "heteroaryl" means an aryl group
wherein at least one of the ring members is a heteroatom, and
preferably 5 or 6 atoms in each ring. The heteroaromatic group
preferably has 1 or 2 oxygen atoms, 1 or 2 sulfur atoms, and/or 1
to 4 nitrogen atoms in the ring, and may be bonded to the remainder
of the molecule through a carbon or heteroatom. Exemplary
heteroaromatics include furyl, thienyl, pyridyl, oxazolyl,
pyrrolyl, indolyl, quinolinyl, or isoquinolinyl and the like.
Exemplary substituents include one or more of the following groups:
hydrocarbonyl, substituted hydrocarbonyl, keto (i.e., .dbd.O),
hydroxy, protected hydroxy, acyl, acyloxy, alkoxy, alkenoxy,
alkynoxy, aryloxy, halogen, amido, amino, nitro, cyano, thiol,
ketals, acetals, esters and ethers.
[0205] As use herein, "cycloalkyl" refers to an alkyl group in
which the end carbon atoms of the alkyl chain are covalently bonded
to one another. The numbers "m" and "n" refer to the number of
carbon atoms in the ring formed. Thus for instance, a (3C-8C)
cycloalkyl group refers to a three, four, five, six, seven or eight
member ring, that is, cyclopropane, cyclobutane, cyclopentane,
cyclohexane, cycloheptane and cyclooctane. A cycloalkyl group of
this invention may optionally be substituted with one or more
fluorine groups and/or one or more alkyl groups.
[0206] As used herein, the term "heterocycloalkyl" means a
cycloalkyl group wherein at least one of the backbone carbon atoms
is replaced with a heteroatom.
[0207] As used herein, the term "alkynyl" refers to an unsaturated,
straight chain hydrocarbon group having from two to ten carbon
atoms therein and in which at least two carbon atoms are bonded
together by a triple bond.
[0208] As used herein, the term "alkenyl" refers to an unsaturated,
straight chain hydrocarbon group having from two to ten carbon
atoms therein and in which at least two carbon atoms are bonded
together by a double bond.
[0209] When a functional group, such as an amine, is termed
"protected," this means that the group is in modified form to
preclude undesired side reactions at the protected site. Suitable
protecting groups for the copolymers of the present disclosure will
be recognized from the present application taking into account the
level of skill in the art, and with reference to standard
textbooks, such as Greene, T. W. et al., Protective Groups in
Organic Synthesis Wiley, New York (1991). Carboxy groups can be
protected as esters thereof, for example methyl, ethyl, tert-butyl,
benzyl, and 4-nitrobenzyl esters. Hydroxy groups can be protected
as ethers or esters thereof, for example methoxymethyl ethers,
tetrahydropyranyl ethers, benzyl ethers, acetates or benzoates.
Mercapto groups can be protected as thioethers or thioesters, for
example pyridyl thioethers, maleimide thioethers, tert-butyl
thioethers, thioacetates or thiobenzoates. Amino groups can be
protected as carbamates, such as tert-butoxycarbonyl derivatives,
or as amides, such as acetamides and benzamides.
[0210] As is well-known in the art, nomenclature of PEG molecular
weight can use the overall molecular weight (including the PEG end
groups) or the number of repeat units. For example PEG.sub.12 is
also known as PEG.sub.0.6kDa or PEG.sub.0.6k. PEG.sub.36 is also
known as PEG.sub.1.6kDa or PEG.sub.1.6k. PEG.sub.48 is also known
as PEG.sub.2.2kDa or PEG.sub.2.2k. A particular form of PEG.sub.48
is also known as PEG.sub.24-amido-PEG.sub.24, but has also been
generally described as PEG.sub.2.2kDa or PEG.sub.2.2k.
[0211] PEGMA.sub.4-5 (Poly(ethylene glycol) methyl ether
methacrylate, average Mn=300) is also known as PEGMA.sub.0.3kDA or
PEGMA.sub.0.3k or PEGMA.sub.300, which is the average molecular
weight of a mixture of PEGMA.sub.4 and PEGMA.sub.5. Similarly,
PEGMA.sub.7-9 (Poly(ethylene glycol) methyl ether methacrylate,
average Mn=500) is also known as PEGMA.sub.0.5kDA or PEGMA.sub.0.5k
or PEGMA.sub.500, which is the average molecular weight of a
mixture of PEG.sub.7 and PEG.sub.9. Similarly, PEGMA.sub.17-19
(Poly(ethylene glycol) methyl ether methacrylate, average Mn=1000)
is also known as PEGMA.sub.1kDA or PEGMA.sub.1k or PEGMA.sub.1000,
which is the average molecular weight of a mixture of PEGMA.sub.17
and PEGMA.sub.19.
[0212] As used herein, a "labile bond" is a covalent bond that is
capable of being selectively broken. That is, the labile bond may
be broken in the presence of other covalent bonds without the
breakage of the other covalent bonds. For example, a disulfide bond
is capable of being broken in the presence of thiols without
cleavage of other bonds, such as carbon-carbon, carbon-oxygen,
carbon-sulfur, carbon-nitrogen bonds, which may also be present in
the molecule. Labile also means "cleavable."
[0213] As used herein, a "labile linkage" is a chemical compound
that contains a labile bond and provides a link or spacer between
two other groups. The groups that are linked may be chosen from
compounds such as biologically active compounds, membrane active
compounds, compounds that inhibit membrane activity, functional
reactive groups, monomers, and cell targeting signals. The spacer
group may contain chemical moieties chosen from a group that
includes alkanes, alkenes, esters, ethers, glycerol, amide,
saccharides, polysaccharides, and heteroatoms such as oxygen,
sulfur, or nitrogen. The spacer may be electronically neutral, may
bear a positive or negative charge, or may bear both positive and
negative charges with an overall charge of neutral, positive or
negative.
[0214] As used herein, "pH-labile" or "pH-sensitive" refers to the
selective breakage of a covalent bond under acidic conditions
(pH<7), or that the covalent bond is broken more rapidly under
acidic conditions (pH<7) than under neutral conditions. That is,
the pH-labile bond may be broken under acidic conditions in the
presence of other covalent bonds that are not broken.
[0215] As used herein, a "micelle" includes a particle comprising a
core and a hydrophilic shell, wherein the core is held together at
least partially, predominantly or substantially through hydrophobic
interactions. In certain instances, as used herein, a "micelle" is
a multi-component, nanoparticle comprising at least two domains,
the inner domain or core, and the outer domain or shell. The core
is at least partially, predominantly or substantially held together
by hydrophobic interactions, and is present in the center of the
micelle. As used herein, the "shell of a micelle" is defined as
non-core portion of the micelle.
[0216] As used herein, a particle or assembly is "micelle-like" if
it substantially behaves like a micelle: (1) it is formed by
spontaneous self association of block copolymers to form organized
assemblies (e.g., micelles) upon dilution from a water-miscible
solvent (such as but not limited to ethanol) to aqueous solvents
(for example phosphate-buffered saline, pH 7.4); (2) it is stable
to dilution (e.g., down to a polymer concentration of 100 .mu.g/ml,
50 .mu.g/ml, 10 .mu.g/ml, 5 ug/ml or 1 .mu.g/ml, which constitutes
the critical stability concentration or the critical micelle
concentration (CMC)); and/or (3) it has an increasing instability
as the concentration of organic solvent increases, such organic
solvents including, but not limited to dimethylformamide (DMF),
dimethylsulfoxide (DMS), and dioxane.
[0217] The term "effective amount," in the context of methods as
described herein for delivering a therapeutic or diagnostic agent
intracellularly by administering to a subject a lipid nanoparticle
and a membrane-destabilizing polymer, refers to an amount the lipid
nanoparticle and an amount of the membrane-destabilizing polymer
that together is sufficient to achieve detectable delivery of the
therapeutic or diagnostic agent to the cytosol of a target cell or
target tissue. Reference herein to delivery of a therapeutic or
diagnostic agent to the "cytosol" includes delivery of such a
therapeutic or diagnostic agent that may ultimately be targeted to
the nucleus of a cell subsequent to its delivery to the
cytosol.
[0218] The term "effective amount" or "therapeutically effective
amount," in the context of treatment of a disease by administering
to a subject a lipid nanoparticle and membrane-destabilizing
polymer as described herein, refers to an amount the lipid
nanoparticle (comprising the therapeutic agent) and an amount of
the membrane-destabilizing polymer that together is sufficient to
inhibit the occurrence or ameliorate one or more symptoms of the
disease in the subject. An effective amount of an agent-containing
lipid nanoparticle and membrane-destabilizing polymer is
administered according to the present methods in an "effective
regime." The term "effective regime" refers to a combination of
agent-containing lipid nanoparticle being administered,
membrane-destabilizing polymer being administered, and dosage
frequency adequate to accomplish treatment or prevention of the
disease.
[0219] The term "patient" or "subject," in the context of
therapeutic or diagnostic agent delivery in vivo as described
herein, includes human and other mammalian subjects.
[0220] Percent sequence identity is determined by conventional
methods. See, e.g., Altschul et al., Bull. Math. Bio. 48:603, 1986,
and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,
1992. For example, two amino acid sequences can be aligned to
optimize the alignment scores using a gap opening penalty of 10, a
gap extension penalty of 1, and the "BLOSUM62" scoring matrix of
Henikoff and Henikoff, supra. The percent identity is then
calculated as: ([Total number of identical matches]/[length of the
longer sequence plus the number of gaps introduced into the longer
sequence in order to align the two sequences])(100). Those skilled
in the art appreciate that there are many established algorithms
available to align two amino acid sequences. The "FASTA" similarity
search algorithm of Pearson and Lipman (Proc. Nat'l Acad. Sci. USA
85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990) is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and a
second amino acid sequence.
[0221] When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to .+-.10%.
BRIEF DESCRIPTION OF THE DRAWINGS
[0222] FIGS. 1A and 1B show reduction in orotic acid (OA) and
plasma ammonia levels in hyperammonemic OTC-spf.sup.ash mice
treated with mRNA encoding ornithine transcarbamylase (OTC).
Hyperammonemia was induced in OTC-spf.sup.ash mice by treatment
with AAV2/8 vector/OTC shRNA, and four days after AAV dosing, mice
were treated twice per week with 1 mg/kg of OTC mRNA formulated in
DOTAP:CHEMS:CHOL:DMPE-PEG.sub.2k (50:32:16:2) at N:P 7+co-injection
of 50 mg/kg P67. See Example 21. Urine collected at day 6 and day
13 (post-AAV treatment) was analyzed for OA levels that were
normalized to creatine levels, and plasma collected at day 13 was
analyzed for ammonia levels. Orotic acid levels are shown in FIG.
1A (black fill=day 6; crosshatch fill=day 13). Plasma ammonia
levels are shown in FIG. 1B.
[0223] FIGS. 2A and 2B show reduction in orotic acid (OA) and
plasma ammonia levels in hyperammonemic OTC-spf.sup.ash mice
treated with mRNA encoding ornithine transcarbamylase (OTC).
Hyperammonemia was induced in OTC-spf.sup.ash mice by treatment
with AAV2/8 vector/OTC shRNA, and four days after AAV dosing, mice
were treated twice per week with 1 mg/kg of OTC mRNA formulated in
DOTAP:CHEMS:CHOL:DSPE-PEG.sub.2k (50:32:8:10) at N:P 7+co-injection
of 35 mg/kg P82. See Example 21. Urine collected at day 6 and day
13 (post-AAV treatment) was analyzed for OA levels that were
normalized to creatine levels, and plasma collected at day 13 was
analyzed for ammonia levels. Orotic acid levels are shown in FIG.
2A (black fill=day 6; crosshatch fill=day 13). Plasma ammonia
levels are shown in FIG. 2B.
[0224] FIG. 3 schematically depicts a proposed mechanism of action
for delivery of an mRNA to the cytosol of a target cell using a
membrane-destabilizing polymer and an LNP carrier in accordance
with an embodiment of the present disclosure. (A) Two separate
nanoparticle solutions are prepared: one nanoparticle containing
the membrane-destabilizing polymer and a second nanoparticle that
is the LNP comprising the mRNA. (B) The two nanoparticle solutions
are then mixed immediately prior to in vivo administration. (C)
While not intending to be bound by theory, it is believed that the
polymer and mRNA/LNP nanoparticles co-localize within the same
intracellular vesicle (e.g., endosome) of the target cell, where
(D) the membrane-destabilizing polymer triggers release of the mRNA
into the cytosol for translation into protein.
DESCRIPTION OF THE INVENTION
[0225] The present invention is directed to methods, compositions,
and delivery systems for in vivo delivery of a therapeutic or
diagnostic agent to the cytosol of a target cell (e.g., in vivo
cytosolic delivery of the agent to a plurality of target cells
within a target tissue). The methods, compositions, and delivery
systems may be used for intracellular delivery of a wide variety of
molecular agents, including polynucleotides, peptide, proteins, and
small molecules, and thus have a variety of diagnostic and
therapeutic applications, including, e.g., the treatment of cancer,
infectious disease, and diseases characterized by protein
deficiencies.
[0226] The present invention relates, inter alia, to formulations
used for delivery of the therapeutic or diagnostic agent.
Generally, the therapeutic or diagnostic agent is formulated in a
lipid nanoparticle ("LNP"; e.g., a liposome) and either a
membrane-destabilizing polymer is added to the formulation (a
co-formulation for co-injection of LNP and polymer) or the LNP
"carrier" formulation and the membrane-destabilizing polymer are
used separately via separate (e.g., sequential) injections into a
subject. Either one or both of the LNP and membrane-destabilizing
polymer may include a targeting ligand that binds to a molecule on
the surface of the desired cell target. In certain other
embodiments, neither the LNP nor the membrane-destabilizing polymer
have a targeting ligand. The function of the lipid nanoparticle is
to encapsulate the therapeutic or diagnostic agent, preventing its
interaction with various components of the systemic circulation and
facilitating delivery to and uptake into the desired tissues and
cells. The lipid nanoparticle may also participate in lysis of
endosomes. While not intending to be bound by theory, it is
believed that the membrane-destabilizing polymer functions as an
agent to elicit or enhance the delivery of the therapeutic or
diagnostic agent into the cytosol of target cells, possibly by
improving endosomal escape of the LNP from the endosome. For
example, the lipid nanoparticle and the membrane-destabilizing
polymer may co-localize to an intracellular vesicle within the
target cell, where the membrane-destabilizing polymer may
facilitate release of the therapeutic or diagnostic agent by
disrupting the vesicle membrane. As shown in the working examples
herein, the combination of LNP and membrane-destabilizing polymer
demonstrated enhanced activity of the delivered agent (either using
co-injection or sequential injections) as compared to the use of
LNPs alone. See Examples 1, 2, 18, and 20, infra. Again without
intending to be bound by theory, this result is believed to be due
to enhanced delivery of the agent into the target cells when
polymer is used in combination with an LNP carrier.
[0227] Accordingly, in one aspect, the present invention provides a
method for delivering a therapeutic or diagnostic agent to the
cytosol of a target cell. The method generally includes
administering to the subject (a) an effective amount of a lipid
nanoparticle comprising the therapeutic or diagnostic agent and (b)
an effective amount of a membrane-destabilizing polymer, where the
therapeutic or diagnostic agent is delivered to the cytosol of the
target cell. In some embodiments of the method, at least one of the
lipid nanoparticle and membrane-destabilizing polymer includes a
first targeting ligand that specifically binds to a molecule on the
surface of the target cell.
[0228] In another aspect, the present invention provides a
composition for delivering a therapeutic or diagnostic agent to the
cytosol of a target cell. The composition generally includes (a) a
lipid nanoparticle comprising the therapeutic or diagnostic agent
and (b) a membrane-destabilizing polymer. In some embodiments of
the composition, at least one of the lipid nanoparticle and
membrane-destabilizing polymer includes a first targeting ligand
that specifically binds to a molecule on the surface of the target
cell. Such compositions may be used in certain embodiments of the
delivery methods described herein, particularly embodiments
comprising co-injection of a membrane-destabilizing polymer and a
lipid nanoparticle comprising the therapeutic or diagnostic
agent.
[0229] In another aspect, the present invention provides a delivery
system for delivering a therapeutic or diagnostic agent to the
cytosol of a target cell. The delivery system generally includes
(a) a carrier composition comprising a lipid nanoparticle, where
the lipid nanoparticle comprises the therapeutic or diagnostic
agent and (b) an enhancer composition comprising a
membrane-destabilizing polymer. In some embodiments of the delivery
system, at least one of the lipid nanoparticle and
membrane-destabilizing polymer includes a first targeting ligand
that specifically binds to a molecule on the surface of the target
cell. Such delivery systems may be used in certain embodiments of
the delivery methods described herein, particularly embodiments
comprising separate (e.g., sequential) injection of a
membrane-destabilizing polymer and a lipid nanoparticle comprising
the therapeutic or diagnostic agent.
[0230] In another aspect, the present invention provides a
membrane-destabilizing polymer as described herein.
[0231] In another aspect, the present invention provides a lipid
nanoparticle as described herein.
[0232] Typically, where a membrane-destabilizing polymer is added
to a lipid nanoparticle formulation in accordance with the present
disclosure (e.g., for making a composition comprising (a) a lipid
nanoparticle comprising a therapeutic or diagnostic agent and (b) a
membrane-destabilizing polymer), the polymer is not contained
within the lipid nanoparticle. In certain embodiments of the
various aspects disclosed herein, the membrane-destabilizing
polymer forms a nanoparticle that is compositionally distinct from
the lipid nanoparticle. For example, where the
membrane-destabilizing polymer is a polymer comprising hydrophilic
and hydrophobic segments, the polymer may form a micelle or
micelle-like particle in aqueous solution.
[0233] A wide variety of therapeutic and diagnostic agents are
generally known and may be used in accordance with the present
methods, compositions, and delivery systems. The therapeutic or
diagnostic agent to be delivered can be, for example, a
polynucleotide, a protein, a peptide, or a small molecule. Suitable
classes of therapeutic agents include, for example, anti-cancer
agents, anti-infective agents (e.g., anti-viral or anti-bacterial
agents), immunomodulatory agents (e.g., immunosuppressive or
immunostimulatory agents), anti-inflammatory agents, or agents that
modulate a cellular metabolic activity. Suitable diagnostic agents
include, e.g., a variety of detectable agents, which may be used
alone or as a conjugate (label) to another molecule (e.g., a
polynucleotide, a protein, a peptide, or a small molecule) having a
desired property useful in a diagnostic method (e.g., a binding
specificity for a desired intracellular target). General classes of
labels that can be used in the present invention include, but are
not limited to, radioactive isotopes, paramagnetic isotopes,
compounds that can be imaged by positron emission tomography (PET),
fluorescent or colored compounds, compounds which can be imaged by
magnetic resonance, chemiluminescent compounds, bioluminescent
compounds, and other imaging reagents.
[0234] Methods for formulating lipid nanoparticles for drug
delivery are generally known in the art and may be adapted for use
in the context of the present invention. For example, lipid
nanoparticle formulations for delivery of small RNAs are discussed
in, e.g., Hong and Nam, Theranostics 4:1211-1232, 2014; Asai and
Oku, Biol. Pharm. Bull. 37:201-205, 2014; and Tam et al.,
Pharmaceutics 5:498-507, 2013. Lipid particle formulations and
lipid design for drug delivery are also discussed in, e.g., Samad
et al., Current Drug Delivery 4:297-305, 2007; Martin et al.,
Current Pharmaceutical Design 11:375-394, 2005; Hafez et al.,
Biophysical Journal 79:1438-1446, 2000; Jayaraman et al., Angew.
Chem. Int. Ed. 51:8529-8533, 2012; Li and Schick, Biophysical
Journal 80:1703-1711, 2001; Adami et al., Molecular Therapy
19:1141-1151, 2011); Dabkowska et al., J. R. Soc. Interface
9:548-561, 2012; Gubernator, Expert Opinion on Drug Delivery
8:565-80, 2011; Whitehead et al., Nat. Commun. 5:4277, 2014; and
Dong et al., Proc. Natl. Acad. Sci. USA 111:3955-60, 2014.
[0235] For LNP formulations comprising a polynucleotide agent, a
lipid nanoparticle includes one or more cationic lipids, which are
useful, inter alia, in complexing with the polynucleotide via
electrostatic interactions. The lipid nanoparticle may further
include additional lipids, which may serve various purposes such as
aiding manufacturing and storage stability as well as modulation of
the biodistribution. Biodistribution may also be modulated by
incorporation of targeting ligands conjugated to the lipids part of
the lipid nanoparticle. Lipid nanoparticles comprising
polynucleotides are typically formulated with a N:P ratio ranging
from about 1 to about 30. In more specific variations, the N:P
ratio is from about 1 to about 14, from 1 to about 7, or from about
3 to about 7 (e.g., an N:P ratio of about 3, about 3.5, or about
7).
[0236] In certain embodiments, a cationic lipid for forming the
lipid nanoparticle comprises a quaternary amine and is consequently
permanently positively charged. Particularly suitable, permanently
charged cationic lipids that may be used in polynucleotide LNP
formulations include, for example,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTMA), N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
chloride (DOTAP), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
(DOEPC), 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (DLEPC),
1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC),
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1),
N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarb-
oxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5),
Dioctadecylamido-glycylspermine (DOGS),
3b-[N--(N',N'-dimethylaminoethyl)carbamoyl]cholesterol (DC-Chol),
Dioctadecyldimethylammonium Bromide (DDAB), Saint lipids such as
SAINT-2, N-methyl-4-(dioleyl)methylpyridinium,
1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide
(DMRIE), 1,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide
(DORIE), 1,2-dioleoyloxypropyl-3-dimethylhydroxyethyl ammonium
chloride (DORI), Di-alkylated Amino Acid (DILA.sup.2) (e.g.,
C18:1-norArg-C16), Dioleyldimethylammonium chloride (DODAC),
1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC),
1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (MOEPC), and
(R)--N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride
(DOTAPen). Also suitable are cationic lipids with headgroups that
are charged at physiological pH, such as primary amines (e.g.,
DODAG N',N'-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide)
and guanidinium head groups (e.g.,
bis-guanidinium-spermidine-cholesterol (BGSC),
bis-guanidiniumtren-cholesterol (BGTC), PONA, and
(R)-5-guanidinopentane-1,2-diyl dioleate hydrochloride (DOPen-G)).
Yet another suitable cationic lipid is
(R)-5-(dimethylamino)pentane-1,2-diyl dioleate hydrochloride
(DODAPen-C1). In certain embodiments, the cationic lipid is a
particular enantiomer or the racemic form, and includes the various
salt forms of a cationic lipid as above (e.g., chloride or
sulfate). For example, in some embodiments, the cationic lipid is
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
(DOTAP-C1) or N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
sulfate (DOTAP-Sulfate).
[0237] In certain variations, a cationic lipid for forming the
lipid nanoparticle utilizes side chains of amino acids as the head
groups, where the .alpha.-amino and .alpha.-carboxyl groups serve
as attachment sites for the hydrophobic tails (also referred to as
a "DiLA.sup.2" architecture; see Adami et al., Molecular Therapy
19:1141-1151, 2011). A particular variant of a cationic lipid
having a DiLA.sup.2 structure is C18:1-norArg-C16. See Adami et
al., supra.
[0238] Typically, a lipid nanoparticle comprising a cationic lipid
as above includes one or more additional lipids. Additional lipids
suitable to be incorporated into the lipid nanoparticles may
include one or more of an anionic lipid, a neutral helper lipid,
and a PEG-conjugated lipid (also referred to herein as a
"PEG-lipid"). Hence in certain embodiments, lipid nanoparticles are
provided that comprise a cationic lipid as above and one or more
additional lipids selected from the group of an anionic lipid, a
helper lipid and a PEG-lipid.
[0239] Anionic lipids for use in cationic lipid-containing LNP
formulations are typically ionizable anionic lipids. While
negatively charged at pH values above the pK.sub.a of the anionic
lipid, an ionizable anionic lipid will generally stabilize other
lipids in the LNP and allow the formation of bilayer vesicles, but
will facilitate fusion of these vesicles as the pH is reduced
toward the pK.sub.a, such as in the acidic endosomal environment of
a cell. Suitable ionizable anionic lipids include cholesteryl
hemisuccinate (CHEMS), phosphatidylserine, palmitoylhomoserine, and
.alpha.-tocopherol hemisuccinate.
[0240] Helper lipids are neutral lipids that help make a stable
liposome dispersion and may also enhance the effectiveness of
cationic lipid-based delivery formulations. Cholesterol (CHOL) is
one particularly suitable helper lipid for used in lipid
nanoparticle formulations. Suitable helper lipids also include
neutral zwitterionic lipids such as, for example,
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),
1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or any related
phosphatidylcholine such as natural sphingomyelin (SM) and
synthetic derivatives thereof such as
1-oleoyl-2-cholesteryl-hemisuccinoyl-sn-glycero-3-phosphocholine
(OChemsPC). Other suitable helper lipids include
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE),
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), and
1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPHyPE).
[0241] In some embodiments, LNPs contain uncharged lipids modified
with hydrophilic polymers such as, e.g., polyethylene glycol (also
referred to herein as "PEG-lipids"). Such PEG-lipids generally
serve to help with assembly of the nanoparticle during its
manufacture, stabilize the lipid nanoparticle, avoid its
aggregation, and prevent its interaction with serum proteins,
opsonins, and RBCs. The polyethylene glycol (PEG) size can vary
from approximately 1 to 5 approximately kDa. Depending on the
relative amounts of these molecules in the formulation and the
length of the hydrocarbon chain, the PEG-lipid can influence the
pharmacokinetic characteristics, biodistribution, and efficacy of a
formulation. PEG-lipids having relatively short lipid hydrocarbon
chains of about 14 carbons dissociate from the LNP in vivo in
plasma with a half-life of less than 1 h. In contrast, a PEG-lipid
with a relatively long lipid hydrocarbon chain length of about 18
carbons circulates fully associated with the formulation for
several days. Hence, in typical embodiments, the PEG-lipid
comprises a lipid hydrocarbon chain of 12 to 20 carbon atoms, 14 to
18 carbon atoms, or of 14 carbon atoms. Typically, the
concentration of the PEG-lipid is about 0.5 to 10 mol %. Examples
of suitable PEG modified lipids include PEGylated ceramide
conjugates and PEGylated distearoylphosphatidyl-ethanolamine
(PEG-DSPE). Other compounds that can be used to stabilize lipid
nanoparticles include gangliosides (GM.sub.t, GM3, and the like).
Preferred PEG-lipids have a PEG size ranging from about 1 to about
5 kDa, with a preferred size range of about 2 to about 5 kDa.
Specific examples are
methoxy-polyethyleneglycol-carbamoyl-dimyristyloxy-propylamine
(PEG2000-c-DMA),
.alpha.-(3'-(1,2-dimyristoyl-3-propanoxy)-carboxamide-propyl]-.omega.-me--
thoxy-polyoxyethylene (PEG2000-c-DOMG),
N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG
2,000), polyethylene gycol-dimyristolglycerol (PEG-DMG), and
N-octanoyl-sphingosine-1-{succinyl[methoxy(polyethylene
glycol)2000]} (C8 PEG2000Ceramide). In some variations of
DMPE-PEG.sub.n where n is 350, 500, 750, 1000 or 2000, the
PEG-lipid is N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG
2,000). In some variations of DSPE-PEG.sub.n where n is 350, 500,
750, 1000 or 2000, the PEG-lipid is
N-(Carbonyl-methoxypolyethyleneglycol
2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG
2,000). In some embodiments, a PEG-lipid is conjugated to a
targeting ligand that specifically binds to molecule on the surface
of a target cell (e.g., an N-acetylgalactosamine (NAG) sugar
residue); such PEG-lipids are particularly useful for formulating
lipid nanoparticles that include a targeting ligand as further
described herein. An exemplary PEG-lipid comprising a NAG moiety is
DSPE-PEG2k-NAG (see, e.g., Examples 19 and 22, infra).
[0242] In certain embodiments, a lipid nanoparticle as above
comprises an ionizable cationic lipid, typically in lieu of any
permanently charged cationic lipid. The ionizable cationic lipid
will have at least one protonatable or deprotonatable group,
typically such that the lipid is positively charged at a pH at or
below physiological pH (e.g., pH 7.4), and neutral at a second pH,
preferably at or above physiological pH. It will be understood that
the addition or removal of protons as a function of pH is an
equilibrium process, and that the reference to a charged or a
neutral lipid refers to the nature of the predominant species and
does not require that all of the lipid be present in the charged or
neutral form. In certain embodiments, ionizable cationic lipids
have a pK.sub.a of the protonatable group in the range of about 4
to about 11. Most preferred is a pK.sub.a of about 4 to about 7,
because these lipids will be cationic at a lower pH formulation
stage, while particles will be largely (though not completely)
surface neutralized at physiological pH around pH 7.4. One of the
benefits of this pK.sub.a is that at least some nucleic acid
associated with the outside surface of the particle will lose its
electrostatic interaction at physiological pH and be removed by
simple dialysis; thus greatly reducing the particle's
susceptibility to clearance. Suitable ionizable cationic lipids for
use in accordance with the present invention include, for example,
Dioctadecyldimethylammonium bromide (DDAB),
1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA),
2,2-dilinoleyl-4-(2dimethylaminoethyl)-[1,3]-dioxolane
(DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (DLin-MC3-DMA),
1,2-Dioleoyloxy-3-dimethylaminopropane (DODAP),
1,2-Dioleyloxy-3-dimethylaminopropane (DODMA),
Morpholinocholesterol (Mo-CHOL), lipidoids such as C12-200 (see
Love et al., Proc. Natl. Acad. Sci. USA 107:1864-9, 2010),
lipopeptide type compounds such as cKK-E12 (Dong et al., Proc.
Natl. Acad. Sci. USA 111:3955-60, 2014), and lipids such as
AIC-0217 and AIC-0218 (Acuitas Therapeutics, Vancouver, BC). Other
suitable ionizable cationic lipids may, for example, be derived
from cationic lipid structures previously described herein.
[0243] In some embodiments, a lipid nanoparticle composition
contains one or more cationic lipids that are from about 0.5% to
about 70% (mol %) of the total amount of lipid and
delivery-enhancing components, including any polymeric (e.g., PEG)
component, but not including the polynucleotide (e.g., RNA)
component. In more particular variations, a lipid nanoparticle
composition contains one or more cationic lipids from about 10% to
about 55%, one or more cationic lipids from about 15% to about 35%,
or one or more cationic lipids from about 35% to about 55%.
[0244] In certain embodiments, a lipid nanoparticle composition
contains one or more non-cationic lipids, where the non-cationic
lipids are from about 2% to about 95% (mol %) of the total amount
of lipid and delivery-enhancing components, including any polymeric
(e.g., PEG) component, but not including the polynucleotide (e.g.,
RNA) component. In some embodiments, a lipid nanoparticle
composition contains one or more non-cationic lipids from about 20%
to about 75%, or from about 45% to about 75%, or from about 45% to
about 55%. In other variations, a lipid nanoparticle composition
contains one or more non-cationic lipids from about 10% to about
50%.
[0245] In some embodiments, a lipid nanoparticle composition
contains one or more polymeric lipids (e.g., PEG-lipid), where the
polymeric lipids are from about 0.2% to about 20% (mol %) of the
total amount of lipid and delivery-enhancing components, including
any polymeric (e.g., PEG) component, but not including the
polynucleotide (e.g., RNA) component. In some embodiments, a lipid
nanoparticle composition contains one or more polymeric lipids from
about 0.5% to about 10%, or one or more polymeric lipids from about
1% to about 5% of the composition.
[0246] Lipid nanoparticle formulations comprising small molecule
agents are also known. See, e.g., Gubernator, Expert Opinion on
Drug Delivery 8:565-80, 2011. For example, small molecules can be
encapsulated in, e.g., a DSPC:CHOL:DSPE-PEG (50:45:5 mol %)
liposome using a passive or an active loading method. Basically,
for a passive loading method, the lipids are solubilized in organic
solvent, then the solvent is evaporated to form a thin lipid film
which is hydrated with an aqueous solution containing a hydrophilic
or hydrophobic drug to be encapsulated. The liposome mixture is
then typically homogenized by vortex and extruded through
polycarbonate membrane in order to reduce the particle size (e.g.,
to .about.100 nm). Non-encapsulated drug can be removed using
dialysis or column filtration.
[0247] Ionizable small molecules can be actively trapped into
liposomes (remote loading method). Typically, in this particular
case, the drug is protonated or precipitated inside the preformed
liposomes thus remaining entrapped in the liposome core. Typically,
a pH gradient (acetate, citrate or ammonium sulfate) where there is
a 1 to 3 pH unit difference between the liposome inner and outer
compartment is used to encapsulate the ionizable small molecules. A
metal gradient (Cu.sup.2+, Mn.sup.2+ or Mg.sup.2+ gradient) can
also be used to actively load a drug into liposomes. Ionophores
such as A23187 can also be used generate a pH gradient in the
liposome using K.sup.+, Mn.sup.2+ or Mg.sup.2+. An EDTA gradient
method can also be used to actively trap small molecules inside a
liposome. In the remote loading method, the liposomes typically are
formed by a simple lipid-film hydration technique (e.g., as
described above for the passive entrapment method with the
exception that the hydration buffer contain the solute required to
generate the gradient across the lipid bilayer). The
non-encapsulated solute is typically removed by dialysis or column
filtration. Following the liposome formation and establishment of a
gradient across the liposomal bilayers, an unprotonated drug is
added in the loading buffer outside the liposome and can cross the
lipid bilayer and becomes protonated inside the liposome, and then
become stabilized by the anions present in the internal aqueous
compartment of the liposome. The suspension may need to be
incubated above the phase transition temperature of the liposomal
lipids to accelerate the drug loading. The non-encapsulated free
drug can be removed, by dialysis or by ion exchange
chromatography.
[0248] Lipid nanoparticle formulations for protein or peptide
therapeutics are also generally known. In some embodiments,
proteinaceous agents are incorporated into liposomes by a lipid
film hydration method. For example, a protein may be incorporated
into PEGylated liposomes composed of, e.g., egg phosphatidylcholine
(EPC), cholesterol, sodium cholesterol-3-sulfate and
distearolyphosphatidyl ethanolamine-N-PEG 2000 (DSPE-PEG [2000]).
Such a formulation method was shown to increase pharmacokinetics
substantially for tPA incorporated into a liposome. See Kim et al.,
Biomaterials 30:5751-5756, 2009.
[0249] In some embodiments, a lipid nanoparticle composition
includes a cationic lipid, an anionic lipid, a helper lipid, and a
PEG-lipid. Such a mixture of LNP lipid components can be
represented by the formula [cationic lipid].sub.w:[anionic
lipid].sub.x:[helper lipid].sub.y: [PEG-lipid].sub.z, where the
subscripts w, x, y, and z represent the mole % of each lipid
component within the mixture (not including the therapeutic or
diagnostic agent component (e.g., polynucleotide) of the LNP). This
formula can be alternatively expressed as [cationic lipid]:[anionic
lipid]:[helper lipid]:[PEG-lipid](w:x:y:z), where w, x, y, and z
represent the mole % of the cationic lipid, anionic lipid, helper
lipid, and PEG-lipid, respectively. In various embodiments, each of
the cationic lipid, anionic lipid, helper lipid, and PEG-lipid are
selected from the exemplary lipids disclosed herein. In some
embodiments, w is from about 10 to about 70, from about 30 to about
60, or from about 35 to about 55; x is from 0 to about 60, from 0
to about 50, from about 10 to about 50, or from about 20 to about
45; y is from about 5 to about 40, from about 5 to about 30, or
from about 5 to about 20; and z is from about 1 to about 20, from
about 2 to about 20, or from about 5 to about 15. For example, a
lipid mixture having the cationic lipid DOTAP present at about 50
mole %, the anionic lipid CHEMS present at about 32 mole %, the
helper lipid CHOL present at about 8 mole %, and the PEG-lipid
DMPE-PEG2k present at about 10 mole % can be expressed as
DOTAP.sub.50:CHEMS.sub.32:CHOL.sub.8:DMPE-PEG2k.sub.10 or as
DOTAP:CHEMS:CHOL:DMPE-PEG2k (50:32:8:10).
[0250] In particular embodiments, a lipid nanoparticle for use in
accordance with the present invention includes a mixture of lipid
components comprising (i) a cationic lipid from about 30 mole % to
about 60 mole %; (ii) an anionic lipid from 0 mole % to about 50
mole %; (iii) a helper lipid from about 1 mole % to about 50 mole
%; and (iv) a PEG-lipid from about 1 mole % to about 20 mole %.
Typically, the cationic lipid is a cationic lipid that is
permanently charged at physiological pH (e.g., DOTAP). If present,
the anionic lipid is typically an ionizable anionic lipid such as,
for example, CHEMS. A particularly suitable helper lipid for use
such embodiments is cholesterol (CHOL), and particularly suitable
PEG-lipids include DSPE-PEG2k and DMPE-PEG2k. An excess of cationic
lipid to anionic lipid, if present, is preferred. In some
variations, (i) the cationic lipid (e.g., DOTAP) is present in the
lipid mixture from about 35 mole % to about 55 mole %, from about
40 mole % to about 55 mole %, from about 45 mole % to about 55 mole
%, or from about 40 mole % to about 50 mole %; (ii) the anionic
lipid (e.g., CHEMS) is present in the lipid mixture from 0 mole %
to about 45 mole %, from about 10 mole % to about 45 mole %, from
about 20 mole % to about 45 mole %, from about 30 mole % to about
45 mole %, or from about 30 mole % to about 40 mole %; (iii) the
helper lipid (e.g., CHOL) is present in the lipid mixture from
about 5 mole % to about 50 mole %, from about 5 mole % to about 40
mole %, from about 5 mole % to about 30 mole %, from about 5 mole %
to about 20 mole %, or from about 5 mole % to about 10 mole %; and
(iv), the PEG-lipid (e.g., DSPE-PEG2k or DMPE-PEG2k) is present in
the lipid mixture from about 1 mole % to about 5 mole %, from about
2 mole % to about 20 mole %, from about 2% mole % to about 15 mole
%, from about 2 mole % to about 10 mole %, from about 5 mole % to
about 20 mole %, from about 5 mole % to about 15 mole %, or from
about 5 mole % to about 10 mole %. In some preferred embodiments,
the PEG-lipid is present in the lipid mixture at a mole % greater
than 5 (e.g., from a mole % greater than 5 to about 20 mole %, to
about 15 mole %, or to about 10 mole %); in some such embodiments,
the PEG-lipid is present at mole % of at least about 6, at least
about 7, at least about 8, at least about 9, or least about 10. In
some embodiments of an LNP composition as above wherein an anionic
lipid is absent, the cationic lipid (e.g., DOTAP) is present in the
lipid mixture from about 35 mole % to about 45 mole %; the helper
lipid (e.g., CHOL) is present in the lipid mixture from about 40
mole % to about 50 mole %; and the PEG-lipid (e.g., DSPE-PEG2k or
DMPE-PEG2k) is present in the lipid mixture from about 5 mole % to
about 15 mole %; in some such embodiments, the molar ratio of
[cationic lipid]:[helper lipid]:[PEG-lipid] is about 40:50:10. In
other embodiments of an LNP composition as above wherein an anionic
lipid is present, the cationic lipid (e.g., DOTAP) is present in
the lipid mixture from about 40 mole % to about 55 mole %; the
anionic lipid (e.g., CHEMS) is present in the lipid mixture from
about 25 mole % to about 40 mole %; the helper lipid (e.g., CHOL)
is present in the lipid mixture from about 5 mole % to about 20
mole %; and the PEG-lipid (e.g., DSPE-PEG2k or DMPE-PEG2k) is
present in the lipid mixture from about 2 mole % to about 15 mole
%, from about 2 mole % to about 10 mole %, or from about 5 mole %
to about 15 mole %; in some such embodiments, the molar ratio of
[cationic lipid]:[anionic lipid]:[helper lipid]:[PEG-lipid] is
about 50:32:16:2 or about 50:32:8:10. In more specific variations,
the LNP composition includes a mixture of lipid components (with
the molar ratio of components specified in parentheses) selected
from (a) DOTAP:CHEMS:CHOL:DMPE-PEG2k (50:32:16:2); (b)
DOTAP:CHEMS:CHOL:DSPE-PEG2k (50:32:8:10); (c)
DOTAP:CHEMS:CHOL:DMPE-PEG2k (50:32:8:10); and (d)
DOTAP:CHOL:DSPE-PEG2k (40:50:10). Mixtures of lipid components as
described above are particularly suitable for lipid nanoparticle
compositions comprising a polynucleotide such as, for example, an
mRNA. LNPs comprising a high PEG-lipid content (for example, a mole
% of greater than 5, such as, e.g., about 10%) represent some
preferred embodiments for polynucleotide (e.g., mRNA) delivery, and
as shown by studies described herein, higher PEG-lipid content was
particularly efficacious in methods for delivery of polynucleotides
to cells in vivo. See, e.g., Example 20.
[0251] In some embodiments, a lipid nanoparticle is less than about
200 nm in size. For example, the lipid nanoparticle may be from
about 30 nm to about 150 nm in size. In certain variations, the
size of the lipid nanoparticle (e.g., between about 30 nm and about
150 nm) facilitates delivery to the liver by an enhanced permeation
and retention effect. The lipid nanoparticle may further include a
targeting ligand to target the particle to a desired tissue. The
lipid nanoparticle may have a positive or negative zeta potential;
in some variations, the zeta potential of the lipid nanoparticle is
substantially neutral.
[0252] In accordance with the present invention, a
membrane-destabilizing polymer is either co-formulated with the
lipid nanoparticle containing the therapeutic or diagnostic agent,
for co-injection into a subject, or is separately formulated for
separate injection (e.g., sequential injection) of the LNP and
membrane-destabilizing polymer. Typically, for co-injection
variations, the lipid nanoparticle and membrane-destabilizing
polymer are initially formulated as separate compositions and then
mixed together into a single composition prior to administration
(typically within one hour prior to administration, more typically
within 30 minutes prior to administration, and preferably within 15
minutes or within five minutes prior to administration). The
membrane-destabilizing polymer elicits a permeability change in a
cellular membrane structure (e.g., an endosomal membrane) so as to
permit macromolecules or biomolecules, or small molecules, to enter
a cell or to exit a cellular vesicle (e.g., an endosome or
lysosome). A variety of membrane-destabilizing polymers are
generally known in the art and may be used in accordance with the
present methods described herein. Known types of
membrane-destabilizing polymers include, for example, copolymers
such as amphipathic copolymers, polycationic or amphipathic
peptides, membrane active toxins, and viral fusogenic peptides.
Certain types of particularly suitable membrane-destabilizing
polymers are described, e.g., in International PCT Application
Publication Nos. WO 2009/140427 and WO 2009/140429, each
incorporated by reference herein in its entirety.
[0253] In some embodiments, a membrane-destabilizing polymer is or
comprises a membrane-destabilizing peptide. In particular
variations, a membrane-destabilizing peptide is selected from
TABLE-US-00001 GALA (e.g., WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID
NO: 15)); truncated GALA (e.g., CAEALAEALAEALAEALA (SEQ ID NO:
16)); melittin (e.g., GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 17) or
CGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 18)); HPH-1 (e.g.,
FIIDIIAFLLMGGFIVYVKNL (SEQ ID NO: 19) or CAAFIIDHAFLLMGGFIVYVKNL
(SEQ ID NO: 20)); sHGP (e.g., CARGWEVLKYWWNLLQY (SEQ ID NO: 21));
bPrPp (e.g., MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (SEQ ID NO: 22)); MAP
(e.g., KLALKLALKALKAALKLA (SEQ ID NO: 23)); PTD4 (e.g., YARAAARQARA
(SEQ ID NO: 24)); Maurocalcine (e.g., GDCLPHLKLCKENKDCCSKKCKRRGTNIE
(SEQ ID NO: 25)); SynB3 (e.g., RRLSYSRRRF (SEQ ID NO: 26)); SynB1
(e.g., RGGRLSYSRRRFSTSTGR (SEQ ID NO: 27)); YTA4 (e.g.,
IAWVKAFIRKLRKGPLG (SEQ ID NO: 28)); YTA2 (e.g., YTAIAWVKAFIRKLRK
(SEQ ID NO: 29)); CADY (e.g., GLWRALWRLLRSLWRLLWRA (SEQ ID NO:
30)); Pep-3 (e.g., KWFETWFTEWPKKRK (SEQ ID NO: 31)); Pep-1 (e.g.,
KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 32)); PepFect (e.g.,
AGYLLGK(eNHa)INLKALAALAKKIL (SEQ ID NO: 33)); PepFect-3 (e.g.,
AGYLLGKINLKALAALAKKIL (SEQ ID NO: 34)); Penetratin (e.g.,
RQIKIWFQNRRMKWKK (SEQ ID NO: 35)); KALA (e.g.,
WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 36)); pVEC (e.g.,
LLIILRRRIRKQAHAHSK (SEQ ID NO: 37)); RVG (e.g.,
YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 38)); MPS (e.g.,
AAVALLPAVLLALLAK (SEQ ID NO: 39)); Transportan (e.g.,
GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 40)); TAT (e.g.,
GRKKRRQRRPPQ (SEQ ID NO: 41)); BMV Gag-(7-25) (e.g.,
KMTRAQRRAAARRNRRWTAR (SEQ ID NO: 42)); hCT(18-32)-k7 (e.g.,
KKRKAPKKKRKFA-KFHTFPQTAIGVGAP (SEQ ID NO: 43)); M1073 (e.g.,
MVTVLFRRLRIRRASGPPRVRV (SEQ ID NO: 44)); EB1 (e.g.,
LIRLWSHLIHIWFQNRRLKWKKK (SEQ ID NO: 45)) and MPG-.beta. (e.g.,
GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 46) or
GALFLAFLAAALSLMGLWSQPKKKRKV (SEQ ID NO: 47)).
[0254] The membrane-destabilizing polymer can be a pH sensitive
polymer having membrane-destabilizing activity at a desired pH. In
some embodiments, membrane-destabilizing polymers (e.g., copolymers
such as block copolymers) provided herein are membrane
destabilizing (e.g., in an aqueous medium) at an endosomal pH. In
some embodiments, the membrane-destabilizing polymers are membrane
destabilizing (e.g., in an aqueous medium) at a pH of about 6.5 or
lower, preferably at a pH ranging from about 5.0 to about 6.5, or
at a pH of about 6.2 or lower, preferably at a pH ranging from
about 5.0 to about 6.2, or at a pH of about 6.0 or lower,
preferably at a pH ranging from about 5.0 to about 6.0.
[0255] Typically, in each case, the membrane-destabilizing polymer
can have membrane destabilizing activity at a desired quantity
(e.g., concentration) of polymer. A membrane-destabilizing
characteristic of a polymer can be determined by suitable assays
known in the art. For example, membrane-destabilizing activity of a
polymer can be determined in an in vitro cell assay such as the red
blood cell hemolysis assay or a liposomal leakage assay. An
endosomolytic polymer activity can be determined in an in vitro
cell assay.
[0256] In general, the membrane-destabilizing polymer is composed
of monomeric residues with particular properties. For example, the
polymer may have amines that are primary, secondary, tertiary, or
quaternary and which drive interactions of the polymer with
membranes. These amines may be permanently charged or have
pK.sub.as ranging from 4 to 14. In particular, these pK.sub.as may
be between 4.5 and 7.5 such that they can undergo acid-base
reactions in endosome. The polymers may also have hydrophobic
groups to further enhance interaction with membranes. The polymer
may also have carboxylic functional groups with pK.sub.as in the
range of 4.0 to 7.5.
[0257] In certain embodiments, a membrane-destabilizing polymer
includes one or more monomeric species selected from anionic,
cationic, hydrophobic, and hydrophilic monomeric residues. Anionic
monomeric residues comprise a species charged or chargeable to an
anion, including a protonatable anionic species. Anionic monomeric
residues can be anionic at an approximately neutral pH of 7.2-7.4.
Cationic monomeric residues comprise a species charged or
chargeable to a cation, including a deprotonatable cationic
species. Cationic monomeric residues can be cationic at an
approximately neutral pH of 7.2-7.4. Hydrophobic monomeric residues
comprise a hydrophobic species. Hydrophilic monomeric residues
comprise a hydrophilic species.
[0258] In some variations, a membrane-destabilizing polymer is or
comprises at least one polymer chain that is hydrophobic. In some
such embodiments, the polymer is or comprises at least one polymer
chain that includes a plurality of anionic monomeric residues. In
this regard, for example, the polymer may be or comprise at least
one polymer chain that includes (i) a plurality of hydrophobic
monomeric residues having a hydrophobic species, and (ii) a
plurality of anionic monomeric residues that are preferably anionic
at approximately neutral pH, and substantially neutral or
non-charged at an endosomal pH or weakly acidic pH.
[0259] In such aforementioned embodiments, the polymer can further
include a plurality of cationic species. Accordingly, for example,
the polymer can be or comprise at least one polymer chain that
includes a plurality of anionic monomeric residues (e.g., having
species that are anionic at about neutral pH), and a plurality of
hydrophobic monomeric residues (e.g., having hydrophobic species),
and optionally a plurality of cationic monomeric residues (e.g.,
having species that are cationic at about neutral pH). In such
embodiments, and as discussed further below, the polymer can be or
comprise at least one polymer chain that is charge modulated, and
preferably charge balanced--being substantially overall neutral in
charge.
[0260] In some embodiments, membrane-destabilizing polymer is a
block copolymer comprising a membrane-destabilizing segment (e.g.,
as a block or region of the polymer). The membrane-destabilizing
segment can comprise a plurality of anionic monomeric residues
(e.g., having species that are anionic at about neutral pH), and a
plurality of hydrophobic monomeric residues (e.g., having
hydrophobic species), and optionally a plurality of cationic
monomeric residues (e.g., having species that are cationic at about
neutral pH). In such embodiments, the segment (e.g., block or
region) can be hydrophobic considered in the aggregate. In such
embodiments, the block copolymer may further comprise a hydrophilic
segment.
[0261] In some embodiments of a block copolymer comprising a
membrane-destabilizing block, the block copolymer includes a first
polymer chain defining a first block A of the copolymer and a
second, membrane-destabilizing polymer chain defining a second
block B of the copolymer. For example, the block copolymer can
comprise a first polymer chain defining a first block A of the
copolymer, which is hydrophilic, and a second polymer chain
defining a second block B of the copolymer that includes (i) a
plurality of hydrophobic monomeric residues and (ii) a plurality of
anionic monomeric residues being anionic at serum physiological pH
and substantially neutral or non-charged at an endosomal pH.
[0262] In some embodiments, the membrane-destabilizing polymer is
or comprises at least one polymer chain that includes a plurality
of anionic monomeric residues, a plurality of hydrophobic monomeric
residues, and optionally a plurality of cationic monomeric residues
in ratios adapted to enhance membrane destabilizing or membrane
destabilizing activity of the polymer chain. For example and
without limitation, in such embodiments at pH 7.4, the ratio of
hydrophobic:(anionic+cationic) species ranges from about 1:2 to
about 3:1, and the ratio of anionic:cationic species ranges from
about 1:0 to about 1:5. In other such embodiments, at pH 7.4, the
ratio of hydrophobic:(anionic+cationic) species ranges from about
1:1 to about 2:1, and the ratio of anionic:cationic species ranges
from about 4:1 to about 1:5.
[0263] In some embodiments, the membrane-destabilizing polymer is
or comprises at least one polymer chain that includes a plurality
of cationic monomeric residues, a plurality of hydrophobic
monomeric residues, and optionally a plurality of anionic monomeric
residues in ratios adapted to enhance membrane destabilizing or
membrane destabilizing activity of the polymer chain. For example
and without limitation, in such embodiments at pH 7.4, the ratio of
hydrophobic:(cationic+anionic) species ranges from about 1:2 to
about 3:1, and the ratio of cationic:anionic species ranges from
about 1:0 to about 1:20. In other such embodiments, at pH 7.4, the
ratio of hydrophobic:(cationic+anionic) species ranges from about
1:1 to about 2:1, and the ratio of cationic:anionic species ranges
from about 1:0 to about 1:5.
[0264] In some embodiments, the membrane-destabilizing polymer is
or comprises at least one polymer chain that includes a plurality
of cationic monomeric residues, and optionally a plurality of
hydrophobic monomeric residues in ratios adapted to enhance
membrane destabilizing or membrane destabilizing activity of the
polymer chain. For example and without limitation, in such
embodiments at pH 7.4, the ratio of hydrophobic:cationic species
ranges from about 0:1 to about 5:1. In other such embodiments, at
pH 7.4, the ratio of hydrophobic:cationic species ranges from about
0:1 to about 2:1.
[0265] Generally, the membrane-destabilizing polymer can be or
comprise at least one polymer chain that is charge modulated, for
example including hydrophobic monomeric residues together with both
anionic monomeric residues and cationic monomeric residues. The
relative ratio of anionic monomeric residues and cationic monomeric
residues can be controlled to achieve a desired overall charge
characteristic. In typical embodiments, for example, such polymer
or polymer chain can be charge balanced--having a substantially
neutral overall charge in an aqueous medium at physiological pH
(e.g., pH 7.2 to 7.4).
[0266] Embodiments comprising a block copolymer, in which at least
one block is or comprises a membrane-destabilizing polymer, such as
a hydrophobic membrane-destabilizing polymer, can comprise one or
more further polymer chains as additional blocks of the block
copolymer. Generally, such further polymer blocks are not narrowly
critical, and can be or comprise a polymer chain which is
hydrophilic, hydrophobic, amphiphilic, and in each case, which is
neutral, anionic or cationic in overall charge characteristics.
[0267] In some embodiments, the membrane-destabilizing polymer is
or comprises a polymer chain that is adapted to facilitate one or
more additional constituent components and/or functional features.
For example, such polymer chain can comprise an end functional
group (e.g., on the alpha end or omega end of the polymer chain)
adapted for covalently linking, directly or indirectly, to a
targeting ligand (affinity reagent) or a shielding agent.
Additionally or alternatively, such polymer chain can comprise one
or more monomeric residues having a pendant functional group
adapted for conjugating to an agent. Such conjugatable monomeric
residues can be effected for covalently linking, directly or
indirectly, to an affinity reagent, a shielding agent, or other
biomolecular agent. Additionally or alternatively, such polymer
chain can comprise one or more monomeric residues having a
shielding species. For example, shielding monomeric residues can be
derived directly from a polymerization reaction which includes
polymerizable monomers comprising a shielding moiety. Shielding
agents include poly ethylene glycol monomers and/or polymers.
Additionally or alternatively, such polymer chain can comprise one
or more monomeric residues having a two or more pendant functional
groups suitable for cross-linking between polymer chains. Such
cross-linking monomeric residues can be a constituent moiety of a
cross-linked polymer or polymer chain, as derived directly from a
polymerization reaction that includes one or more polymerizable
monomers comprising a multi-functional (e.g., bis-functional)
cross-linking monomer.
[0268] Generally, one or more blocks of a block copolymer can be a
random copolymer block which comprises two or more compositionally
distinct monomeric residues.
[0269] Generally, a single monomeric residue can include multiple
moieties having different functionality--e.g., can comprise
hydrophobic species as well as anionic species, can comprise
hydrophobic species as well as cationic species, or can comprise
anionic species as well as cationic species. Hence, in any
embodiment, the polymer can be or can comprise a polymer comprising
a monomeric residue such as an anionic hydrophobic monomeric
residue--which includes hydrophobic species and anionic species
(e.g., species that are anionic at about neutral pH).
[0270] In typical variations, anionic monomeric residues comprise a
protonatable anionic species. Considered in the aggregate, as
incorporated into a polymer chain, such anionic monomeric residues
can be substantially anionic at a pH of or greater than 7.0 and
substantially neutral (non-charged) at pH of or less than 6.0.
Preferably, such anionic monomeric residues have a pK.sub.a ranging
from about 4 to about 6.8, (e.g., from about 4 to about 6, from
about 4 to about 5, from about 5 to about 6, from about 5 to about
6.8, or from about 5.5 to about 6.8). Anionic monomeric residues
can independently comprise a plurality of monomeric residues having
a protonatable anionic species selected from carboxylic acid,
sulfonamide, boronic acid, sulfonic acid, sulfinic acid, sulfuric
acid, phosphoric acid, phosphinic acid, and phosphorous acid
groups, and combinations thereof. Particularly suitable anionic
monomeric residues may be derived from polymerization of a
(C.sub.2-C.sub.8) alkylacrylic acid.
[0271] Hydrophobic monomeric residues can be charged or noncharged,
generally. Some embodiments include neutral (non-charged)
hydrophobic monomeric residues. In some embodiments, polymer chains
can independently comprise a plurality of monomeric residues having
a hydrophobic species selected from (C.sub.1-C.sub.18) alkyl (e.g.,
(C.sub.2-C.sub.8) alkyl), (C.sub.1-C.sub.18) alkenyl (e.g.,
(C.sub.2-C.sub.8) alkenyl), (C.sub.1-C.sub.18) alkynyl (e.g.,
(C.sub.2-C.sub.8) alkynyl), aryl, heteroaryl, and cholesterol (each
of which may be optionally substituted). In certain embodiments,
the plurality of monomeric residues can be derived from
polymerization of (C.sub.1-C.sub.18) alkyl-ethacrylate (e.g.,
(C.sub.2-C.sub.8) alkyl-ethacrylate), a (C.sub.1-C.sub.18)
alkyl-methacrylate (e.g., (C.sub.2-C.sub.8) alkyl-methacrylate), or
a (C.sub.1-C.sub.18) alkyl-acrylate (e.g., (C.sub.2-C.sub.8)
alkyl-acrylate) (each of which may be optionally substituted).
[0272] Cationic monomeric residues can preferably comprise a
deprotonatable cationic species. Considered in the aggregate, as
incorporated into a polymer chain, such cationic monomeric residues
can be substantially cationic at a pH of or greater than 7.0.
Preferably, such cationic monomeric residues have a pK.sub.a
ranging from about 5.5 to about 9.0 (e.g., from about 6.5 to about
9.0). Cationic monomeric residues can independently comprise a
plurality of monomeric residues having a deprotonatable cationic
species selected from the group consisting of acyclic amine,
acyclic imine, cyclic amine, cyclic imine, and nitrogen-containing
heteroaryl. Preferred cationic monomeric residues can be derived
from polymerization of, in each case optionally substituted,
(N,N-di(C.sub.1-C.sub.6)alkyl-amino(C.sub.1-C.sub.6)alkyl-ethacrylate,
N,N-di(C.sub.1-C.sub.6)alkyl-amino(C.sub.1-C.sub.6)alkyl-methacrylate,
or N,N-di(C.sub.1-C.sub.6)alkyl-amino
(C.sub.1-C.sub.6)alkyl-acrylate.
[0273] In some embodiments, a pH-sensitive membrane-destabilizing
polymer includes a random copolymer chain, such as, e.g., a random
copolymer chain comprising two or more monomeric residue species as
described above. For example, in particular variations, the random
copolymer chain has monomeric residues derived from polymerization
of propyl acrylic acid, N,N-dimethylaminoethylmethacrylate, and
butyl methacrylate. In particular embodiments, the pH-sensitive
polymer is a block copolymer comprising the random copolymer chain
as a membrane-destabilizing polymer block, and further comprising
one or more additional blocks (e.g., a hydrophilic block). For
example, in some embodiments, the polymer is a diblock copolymer
comprising a membrane-destabilizing random copolymer block and a
second block, which can be represented by the schematic
[A].sub.v-[B].sub.w, where [B] represents the
membrane-destabilizing block, [A] represents the second block
(e.g., a hydrophilic block or an amphiphilic block), and the
letters v and w represent the molecular weight (number average) of
the respective blocks in the copolymer. In certain variations of a
block copolymer comprising a membrane-destabilizing polymer block
and a hydrophilic block, the hydrophilic block is polymerized from
both hydrophilic monomers and hydrophobic monomers such that there
are more hydrophilic monomeric residues than hydrophobic monomeric
residues in the hydrophilic block.
[0274] In some variations, a pH-sensitive membrane-destabilizing
polymer is a diblock copolymer having a hydrophilic random
copolymer block and a hydrophobic random copolymer block, where (i)
the hydrophilic block is an amphiphilic block comprising both
hydrophilic monomeric residues and hydrophobic monomeric residues,
where the number of hydrophilic monomeric residues in the
hydrophilic block is greater than the number of hydrophobic
monomeric residues, (ii) the hydrophobic block is an amphiphilic,
membrane-destabilizing block comprising both hydrophobic monomeric
residues and hydrophilic monomeric residues and having an overall
hydrophobic character at a pH of about 7.4, and (iii) each of the
hydrophilic monomeric residues of the hydrophilic and hydrophobic
blocks is independently selected from monomeric residues that are
ionic at a pH of about 7.4, monomeric residues that are neutral at
a pH of about 7.4, and monomeric residues that are zwitterionic at
a pH of about 7.4. In some such embodiments, the monomers used to
prepare the diblock copolymer comprise acrylate(s),
methacrylate(s), acrylamide(s), and/or methacrylamide(s). In
particular variations, the hydrophilic block comprises hydrophilic
monomeric residues that are neutral at a pH of about 7.4, and/or
the hydrophobic block comprises both hydrophilic monomeric residues
that are cationic at a pH of about 7.4 and hydrophilic monomeric
residues that are anionic at a pH of about 7.4. Suitable
hydrophilic and hydrophobic monomeric residues for use in a diblock
copolymer as above are further described herein. In some
embodiments, a diblock copolymer as above is a random block
copolymer of formula I as set forth herein.
[0275] In some variations, a pH-sensitive membrane-destabilizing
polymer is a diblock copolymer having a hydrophilic random
copolymer block and a hydrophobic random copolymer block, where (i)
the hydrophilic block is an amphiphilic block comprising both
hydrophilic monomeric residues and hydrophobic monomeric residues
and having an overall hydrophilic character at a pH of about 7.4,
(ii) the hydrophobic block is an amphiphilic,
membrane-destabilizing block comprising both hydrophobic monomeric
residues and hydrophilic monomeric residues and having an overall
hydrophobic character at a pH of about 7.4, and (iii) each of the
hydrophilic monomeric residues of the hydrophilic and hydrophobic
blocks is independently selected from monomeric residues that are
ionic at a pH of about 7.4, monomeric residues that are neutral at
a pH of about 7.4, and monomeric residues that are zwitterionic at
a pH of about 7.4. In some such embodiments, the monomers used to
prepare the diblock copolymer comprise acrylate(s),
methacrylate(s), acrylamide(s), and/or methacrylamide(s).
[0276] In certain embodiments, a pH-sensitive polymer is covalently
linked to a membrane-destabilizing peptide. For example, the
pH-sensitive polymer may include a plurality of pendant linking
groups, and a plurality of membrane-destabilizing peptides may be
linked to the pH-sensitive polymer via the plurality of pendant
linking groups. In some variations, a peptide comprising a cysteine
residue at either the amino or carboxyl terminus is conjugated to a
monomer containing a disulfide moiety through the cysteine thiol to
form a disulfide bridge. Exemplary membrane-destabilizing peptides
that may be linked to a polymer include, for example,
TABLE-US-00002 GALA (e.g., WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID
NO: 15)); truncated GALA (e.g., CAEALAEALAEALAEALA (SEQ ID NO:
16)); melittin (e.g., GIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 17) or
CGIGAVLKVLTTGLPALISWIKRKRQQ (SEQ ID NO: 18)); HPH-1 (e.g.,
FIIDIIAFLLMGGFIVYVKNL (SEQ ID NO: 19) or CAAFIIDHAFLLMGGFIVYVKNL
(SEQ ID NO: 20)); sHGP (e.g., CARGWEVLKYWWNLLQY (SEQ ID NO: 21));
bPrPp (e.g., MVKSKIGSWILVLFVAMWSDVGLCKKRPKP (SEQ ID NO: 22)); MAP
(e.g., KLALKLALKALKAALKLA (SEQ ID NO: 23)); PTD4 (e.g., YARAAARQARA
(SEQ ID NO: 24)); Maurocalcine (e.g., GDCLPHLKLCKENKDCCSKKCKRRGTNIE
(SEQ ID NO: 25)); SynB3 (e.g., RRLSYSRRRF (SEQ ID NO: 26)); SynB1
(e.g., RGGRLSYSRRRFSTSTGR (SEQ ID NO: 27)); YTA4 (e.g.,
IAWVKAFIRKLRKGPLG (SEQ ID NO: 28)); YTA2 (e.g., YTAIAWVKAFIRKLRK
(SEQ ID NO: 29)); CADY (e.g., GLWRALWRLLRSLWRLLWRA (SEQ ID NO:
30)); Pep-3 (e.g., KWFETWFTEWPKKRK (SEQ ID NO: 31)); Pep-1 (e.g.,
KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 32)); PepFect (e.g.,
AGYLLGK(eNHa)INLKALAALAKKIL (SEQ ID NO: 33)); PepFect-3 (e.g.,
AGYLLGKINLKALAALAKKIL (SEQ ID NO: 34)); Penetratin (e.g.,
RQIKIWFQNRRMKWKK (SEQ ID NO: 35)); KALA (e.g.,
WEAKLAKALAKALAKHLAKALAKALKACEA (SEQ ID NO: 36)); pVEC (e.g.,
LLIILRRRIRKQAHAHSK (SEQ ID NO: 37)); RVG (e.g.,
YTIWMPENPRPGTPCDIFTNSRGKRASNG (SEQ ID NO: 38)); MPS (e.g.,
AAVALLPAVLLALLAK (SEQ ID NO: 39)); Transportan (e.g.,
GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 40)); TAT (e.g.,
GRKKRRQRRPPQ (SEQ ID NO: 41)); BMV Gag-(7-25) (e.g.,
KMTRAQRRAAARRNRRWTAR (SEQ ID NO: 42)); hCT(18-32)-k7 (e.g.,
KKRKAPKKKRKFA-KFHTFPQTAIGVGAP (SEQ ID NO: 43)); M1073 (e.g.,
MVTVLFRRLRIRRASGPPRVRV (SEQ ID NO: 44)); EB1 (e.g.,
LIRLWSHLIHIWFQNRRLKWKKK (SEQ ID NO: 45)) and MPG-.beta. (e.g.,
GALFLGFLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 46) or
GALFLAFLAAALSLMGLWSQPKKKRKV (SEQ ID NO: 47)).
[0277] In some embodiments, a pH-sensitive polymer includes a
random block copolymer of formula I:
##STR00003## [0278] where [0279] A.sub.0, A.sub.1, A.sub.2,
A.sub.3, A.sub.4 and A.sub.5 are each independently selected from
the group consisting of --C--C--, --C(O)(C).sub.aC(O)O--,
--O(C).sub.aC(O)--, --O(C).sub.b--, and --CR.sub.8--CR.sub.9; where
tetravalent carbon atoms of A.sub.0-A.sub.5 that are not fully
substituted with R.sub.1-R.sub.6 and Y.sub.0-Y.sub.5 are completed
with an appropriate number of hydrogen atoms; wherein a and b are
each independently 1-4; and where R.sub.8 and R.sub.9 are each
independently selected from the group consisting of --C(O)OH,
--C(O)Oalkyl, and --C(O)NR.sub.10, where R.sub.8 and R.sub.9 are
optionally covalently linked together to form a ring structure
(e.g., a cyclic anhydride or cyclic imide); [0280] Y.sub.5 is
hydrogen or is selected from the group consisting of
-(1C-10C)alkyl, -(3C-6C)cycloalkyl, --O-(1C-10C)alkyl,
--C(O)O(1C-10C)alkyl, --C(O)NR.sub.11(1C-10C)alkyl, and
-(6C-10C)aryl, any of which is optionally substituted with one or
more fluorine atoms; [0281] Y.sub.0, Y.sub.3, and Y.sub.4 are each
independently selected from the group consisting of a covalent
bond, -(1C-10C)alkyl-, --C(O)O(2C-10C)alkyl-,
--OC(O)(1C-10C)alkyl-, --O(2C-10C)alkyl-, --S(2C-10C)alkyl-, and
--C(O)NR.sub.12(2C-10C)alkyl-; [0282] Y.sub.1 and Y.sub.2 are each
independently selected from the group consisting of a covalent
bond, -(1C-18C)alkyl-, -(3C-18C)branched alkyl,
--C(O)O(2C-18C)alkyl-, --C(O)O(2C-18C)branched alkyl,
--OC(O)(1C-18C)alkyl-, --OC(O)(1C-18C)branched alkyl-,
--O(2C-18C)alkyl-, --O(2C-18C)branched alkyl-, --S(2C-18C)alkyl-,
--S(2C-18C)branched alkyl-, --C(O)NR.sub.12(2C-18C)alkyl-, and
--C(O)NR.sub.12(2C-18C)branched alkyl-, where any alkyl or branched
alkyl group of Y.sub.1 or Y.sub.2 is optionally substituted with
one or more fluorine atoms; [0283] R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.8, R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently hydrogen, --CN, or selected
from the group consisting of alkyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl and heteroaryl, any of which is
optionally substituted with one or more fluorine atoms; [0284]
Q.sub.0 is a residue selected from the group consisting of residues
which are hydrophilic at physiologic pH;
O--[(C).sub.2-3--O].sub.x--R.sub.7; and
O--[(C).sub.2-3--O].sub.x--C(O)--NR.sub.13R.sub.14; where x is
1-48; R.sub.7 is --CH.sub.3 or --CO.sub.2H; and R.sub.13 and
R.sub.14 are each independently hydrogen, --CN, or selected from
the group consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; [0285] Q.sub.1 and
Q.sub.2 are each independently absent or selected from a residue
which is hydrophilic at normal physiological pH; a conjugatable or
functionalizable residue; a residue which is hydrophobic at normal
physiological pH; an alkyl group optionally substituted with one or
more fluorine atoms; and a branched alkyl group optionally
substituted with one or more fluorine atoms; [0286] Q.sub.3 is a
residue which is positively charged at normal physiological pH;
[0287] Q.sub.4 is a residue which is negatively charged at normal
physiological pH, but undergoes protonation at lower pH; [0288] m
is a mole fraction of greater than 0 to 1.0; [0289] n is a mole
fraction of 0 to less than 1.0; [0290] p is a mole fraction of 0 to
less than 1.0; wherein m+n+p=1; [0291] q is a mole fraction of 0.1
to 0.9; [0292] r is a mole fraction of 0.05 to 0.9; [0293] s is
present up to a mole fraction of 0.85; wherein q+r+s=1; [0294] v is
from 1 to 25 kDa; and [0295] w is from 1 to 50 kDa.
[0296] In certain embodiments of a polymer of formula I as above, m
is greater than n+p. In some such variations, p is 0.
[0297] In certain embodiments of a polymer of formula I as above, n
is greater than 0. Particularly suitable polymers of formula I
where n is greater than 0 include polymers where
R.sub.2-A.sub.1-Y.sub.1-Q.sub.1 taken together is a monomeric
residue having an overall hydrophobic character. In some such
variations, the hydrophobic monomer contains an alkyl or branched
alkyl group substituted with one or more fluorine atoms (e.g., at
least one of Y.sub.1 and Q.sub.1 contains the alkyl or branched
alkyl group as specified in formula I for Y.sub.1 and Q.sub.1, and
where the alkyl or branched alkyl group is substituted with the one
or more fluorine atoms).
[0298] In some variations of a polymer of formula I where n is
greater than 0, p is 0. In some such embodiments, m is greater than
n. For example, m is typically greater than n where
R.sub.2-A.sub.1-Y.sub.1-Q.sub.1 taken together is a monomeric
residue having an overall hydrophobic character.
[0299] In some specific embodiments of a polymer of formula I, the
ratio of w:v ranges from about 1:1 to about 5:1, or from about 1:1
to about 2:1.
[0300] Exemplary but non-limiting membrane-destabilizing polymers
can be or comprise a polymer chain which is a random copolymer
represented as formula 1, optionally with one or more
counterions.
[0301] In certain embodiments, the constitutional units of the
second block of formula 1 are derived from the polymerizable
monomers N,N-dimethylaminoethylmethacrylate (DMAEMA), propylacrylic
acid (PAA) and butyl methacrylate (BMA).
[0302] In certain embodiments comprising a pH-sensitive polymer of
formula I, the pH-sensitive polymer is a polymer of formula II:
T1-L-[PEGMA.sub.m-PDSMA.sub.n-BPAM.sub.p].sub.v-[DMAEMA.sub.q-PAA.sub.r--
BMA.sub.s].sub.w II [0303] where [0304] PEGMA is polyethyleneglycol
methacrylate residue with 2-20 ethylene glycol units; [0305] PDSMA
is pyridyl disulfide methacrylate residue; [0306] BPAM is 2-[2-Boc
amino ethoxy] ethyl methacrylate residue; [0307] BMA is butyl
methacrylate residue; [0308] PAA is propyl acrylic acid residue;
[0309] DMAEMA is dimethylaminoethyl methacrylate residue; [0310] m
is a mole fraction of 0.6 to 1; [0311] n is a mole fraction of 0 to
0.4 (e.g., 0 to 0.2); [0312] p is a mole fraction of 0 to 0.4
(e.g., 0 to 0.2); [0313] m+n+p=1; [0314] q is a mole fraction of
0.2 to 0.75; [0315] r is a mole fraction of 0.05 to 0.6; [0316] s
is a mole fraction of 0.2 to 0.75; [0317] q+r+s=1; [0318] v is 1 to
25 kDa; [0319] w is 1 to 25 kDa; [0320] T1 is absent or is the
first targeting ligand; and [0321] L is absent or is a linking
moiety.
[0322] In other embodiments comprising a pH-sensitive polymer of
formula I, the pH-sensitive polymer is a polymer of formula V:
T1-L-[PEGMA.sub.m-M2n].sub.v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub.w
V [0323] where [0324] PEGMA is polyethyleneglycol methacrylate
residue with 2-20 ethylene glycol units; [0325] M2 is a
methacrylate residue selected from the group consisting of [0326] a
(C4-C18)alkyl-methacrylate residue; [0327] a (C4-C18)branched
alkyl-methacrylate residue; [0328] a cholesteryl methacrylate
residue; [0329] a (C4-C18)alkyl-methacrylate residue substituted
with one or more fluorine atoms; and [0330] a (C4-C18)branched
alkyl-methacrylate residue substituted with one or more fluorine
atoms; [0331] BMA is butyl methacrylate residue; [0332] PAA is
propyl acrylic acid residue; [0333] DMAEMA is dimethylaminoethyl
methacrylate residue; [0334] m and n are each a mole fraction
greater than 0, wherein m is greater than n and m+n=1; [0335] q is
a mole fraction of 0.2 to 0.75; [0336] r is a mole fraction of 0.05
to 0.6; [0337] s is a mole fraction of 0.2 to 0.75; [0338] q+r+s=1;
[0339] v is 1 to 25 kDa; [0340] w is 1 to 25 kDa; [0341] T1 is
absent or is the first targeting ligand; and [0342] L is absent or
is a linking moiety.
[0343] Particularly suitable M2 methacrylate residues for use in a
polymer of formula V include 2,2,3,3,4,4,4-heptafluorobutyl
methacrylate residue;
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl
2-methylacrylate residue; 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue (also referred to as 2-propenoic acid,
2-methyl-, 3,3,4,4,5,5,6,6,6-nonafluorohexyl ester residue);
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue;
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue; 2-ethylhexyl methacrylate residue; butyl
methacrylate residue; hexyl methacrylate residue; octyl
methacrylate residue; n-decyl methacrylate residue; lauryl
methacrylate residue; myristyl methacrylate residue; stearyl
methacrylate residue; cholesteryl methacrylate residue; ethylene
glycol phenyl ether methacrylate residue; 2-propenoic acid,
2-methyl-, 2-phenylethyl ester residue; 2-propenoic acid,
2-methyl-, 2-[[(1,1-dimethylethoxy)carbonyl]amino]ethyl ester
residue; 2-propenoic acid, 2-methyl-, 2-(1H-imidazol-1-yl)ethyl
ester residue; 2-propenoic acid, 2-methyl-, cyclohexyl ester
residue; 2-propenoic acid, 2-methyl-,
2-[bis(1-methylethyl)amino]ethyl ester residue; 2-propenoic acid,
2-methyl-, 3-methylbutyl ester residue; neopentyl methacrylate
residue; tert-butyl methacrylate residue; 3,3,5-trimethyl
cyclohexyl methacrylate residue; 2-hydroxypropyl methacrylate
residue; 5-nonyl methacrylate residue; 2-butyl-1-octyl methacrylate
residue; 2-hexyl-1-decyl methacrylate residue; and 2-(tert-butyl
amino)ethyl methacrylate residue.
[0344] In particular variations of a pH-sensitive polymer of
formula II or formula V, PEGMA has 4-5 ethylene glycol units or 7-8
ethylene glycol units. In some embodiments, T1 and L are present.
T1 may include, for example, an N-acetylgalactosamine (NAG)
residue, such as, e.g., a tri-NAG moiety as described further
herein. L may be a hydrophilic moiety such as, for example, a
moiety comprising one or more PEG chains. In some embodiments, L is
a hydrophilic moiety comprising from 2 to 240 ethylene glycol units
(e.g., a polyethylene glycol (PEG) moiety having 2-20 ethylene
glycol units).
[0345] In specific embodiments, a pH-sensitive polymer of formula
II is selected from the group consisting of
NAG-PEG.sub.2-[PEGMA300.sub.m-PDSMA.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub.-
s].sub.w IIa
NAG-PEG.sub.12-[PEGMA300.sub.m-PDSMA.sub.n-BPAM.sub.p].sub.v-[D.sub.q-P.-
sub.r-B.sub.s].sub.w IIb
where "D" is DMAEMA as defined above for formula II, "P" is PAA as
defined above for formula II, "B" is BMA as defined above for
formula II, "NAG" is an N-acetylgalactosamine residue, "PEG.sub.12"
is polyethylene glycol having 12 ethylene glycol units and
functionalized at each end for attachment to the NAG residue and
chain transfer agent, "PEGMA," "PDSMA," and "BPAM" are as defined
above for formula II, and the values for m, n, p, q, r, s, v, and w
are as defined above for formula II. In particular variations of a
polymer of formula IIa, m is from 0.85 to 0.9, n is from 0.1 to
0.15, q is from 0.33 to 0.37, r is from 0.07 to 0.15, s is from
0.52 to 0.57, v is from 3 kDa to 4.5 kDa, and/or w is from 5.5 kDa
to 7 kDa. In particular variations of a polymer of formula IIb, m
is from 0.75 to 0.8, n is from 0.1 to 0.13, p is from 0.1 to 0.12,
q is from 0.25 to 0.37, r is from 0.07 to 0.25, s is from 0.5 to
0.57, v is from 3 kDa to 4.5 kDa, and w is from 5.5 kDa to 7 kDa.
In some specific embodiments, the ratio of w:v ranges from about
1:1 to about 5:1, or from about 1:1 to about 2:1.
[0346] In specific embodiments, a pH-sensitive polymer of formula V
is selected from the group consisting of
NAG-PEG.sub.12-[PEGMA300.sub.m-(F1-BMA).sub.n].sub.v-[D.sub.q-P.sub.r-B.-
sub.s].sub.w Vb
NAG-PEG.sub.12-[PEGMA300.sub.m-(OFl-5TFM-HMA).sub.n].sub.v-[D.sub.q-P.su-
b.r-B.sub.s].sub.w Vc
NAG-PEG.sub.12-[PEGMA300.sub.m-(Fl15-OMA.sub.n)].sub.v-[D.sub.q-P.sub.r--
B.sub.s].sub.w Vd
NAG-PEG.sub.12-[PEGMA300.sub.m-(B-Fl-HMA).sub.n].sub.v-[D.sub.q-P.sub.r--
B.sub.s].sub.w Ve
NAG-PEG.sub.12-[PEGMA300.sub.m-(B-Fl-OMA).sub.n].sub.v-[D.sub.q-P.sub.r--
B.sub.s].sub.w Vf
NAG-PEG.sub.12-[PEGMA300.sub.m-EHMA.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub.-
s].sub.w Vg
NAG-PEG.sub.2-[PEGMA300.sub.m-B.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub.s].s-
ub.w Vh
NAG-PEG.sub.2-[PEGMA300.sub.m-HMA.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub.s]-
.sub.w Vi
NAG-PEG.sub.2-[PEGMA300.sub.m-C8MA.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub.s-
].sub.w Vj
NAG-PEG.sub.12-[PEGMA300.sub.m-C12MA.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub-
.s].sub.w Vk
NAG-PEG.sub.12-[PEGMA300.sub.m-Bu1-OMA.sub.n].sub.v-[D.sub.q-P.sub.r-B.s-
ub.s].sub.w VI
NAG-PEG.sub.12-[PEGMA300.sub.m-NMA.sub.n].sub.v-[D.sub.q-P.sub.r-B.sub.s-
].sub.w Vm
where "D" is DMAEMA as defined above for formula V, "P" is PAA as
defined above for formula V, "B" is BMA as defined above for
formula V, "NAG" is an N-acetylgalactosamine residue, "PEG.sub.12"
is polyethylene glycol having 12 ethylene glycol units and
functionalized at each end for attachment to the NAG residue and
chain transfer agent, "PEGMA" is as defined above for formula V,
"Fl-BMA" is 2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue,
"OFl-5TFM-HMA" is
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue, "F115-OMA" is
2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl 2-methylacrylate
residue, "B-Fl-HMA" is 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue, "B-Fl-OMA" is
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl methacrylate residue,
"EHMA" is 2-ethylhexyl methacrylate residue, "HMA" is hexyl
methacrylate residue, "C8MA" is octyl methacrylate residue, "C12MA"
is lauryl methacrylate residue, "2-Bu1-OMA" is 2-butyl-1-octyl
methacrylate residue, "5-NMA" is 5-nonyl methacrylate residue, and
the values for m, n, q, r, s, v, and w are as defined above for
formula V.
[0347] In some embodiments, the pH-sensitive,
membrane-destabilizing polymer comprises a random block copolymer
of formula Ia:
##STR00004## [0348] where [0349] A.sub.0, A.sub.1, A.sub.2,
A.sub.3, A.sub.4 and A.sub.5 are each independently selected from
the group consisting of --C--C--, --C(O)(C).sub.aC(O)O--,
--O(C).sub.aC(O)--, --O(C).sub.b--, and --CR.sub.8--CR.sub.9--;
where tetravalent carbon atoms of A.sub.0-A.sub.5 that are not
fully substituted with R.sub.1-R.sub.6 and Y.sub.0-Y.sub.5 are
completed with an appropriate number of hydrogen atoms; wherein a
and b are each independently 1-4; and where R.sub.8 and R.sub.9 are
each independently selected from the group consisting of --C(O)OH,
--C(O)Oalkyl, and --C(O)NR.sub.10, where R.sub.8 and R.sub.9 are
optionally covalently linked together to form a ring structure;
[0350] Y.sub.5 is hydrogen or is selected from the group consisting
of -(1C-10C)alkyl, -(3C-6C)cycloalkyl, --O-(1C-10C)alkyl,
--C(O)O(1C-10C)alkyl, --C(O)NR.sub.11(1C-10C)alkyl, and
-(6C-10C)aryl, any of which is optionally substituted with one or
more fluorine atoms; [0351] Y.sub.0, Y.sub.3, and Y.sub.4 are each
independently selected from the group consisting of a covalent
bond, -(1C-10C)alkyl-, --C(O)O(2C-10C)alkyl-,
--OC(O)(1C-10C)alkyl-, --O(2C-10C)alkyl-, --S(2C-10C)alkyl-, and
--C(O)NR.sub.12(2C-10C) alkyl-; [0352] Y.sub.1 and Y.sub.2 are each
independently selected from the group consisting of a covalent
bond, -(1C-18C)alkyl-, -(3C-18C)branched alkyl,
--C(O)O(2C-18C)alkyl-, --C(O)O(2C-18C)branched alkyl,
--OC(O)(1C-18C)alkyl-, --OC(O)(1C-18C)branched alkyl-,
--O(2C-18C)alkyl-, --O(2C-18C)branched alkyl-, --S(2C-18C)alkyl-,
--S(2C-18C)branched alkyl-, --C(O)NR.sub.12(2C-18C)alkyl-, and
--C(O)NR.sub.12(2C-18C)branched alkyl-, where any alkyl or branched
alkyl group of Y.sub.1 or Y.sub.2 is optionally substituted with
one or more fluorine atoms; [0353] R.sub.1, R.sub.2, R.sub.3,
R.sub.4, R.sub.5, R.sub.6, R.sub.8, R.sub.9, R.sub.10, R.sub.11,
and R.sub.12 are each independently hydrogen, --CN, or selected
from the group consisting of alkyl, alkynyl, heteroalkyl,
cycloalkyl, heterocycloalkyl, aryl and heteroaryl, any of which is
optionally substituted with one or more fluorine atoms; [0354]
Q.sub.0 is a residue selected from the group consisting of residues
which are hydrophilic at physiologic pH;
O--[(C).sub.2-3--O].sub.x--R.sub.7; and
O--[(C).sub.2-3--O].sub.x--C(O)--NR.sub.13R.sub.14; where x is
1-48; R.sub.7 is --CH.sub.3 or --CO.sub.2H; and R.sub.13 and
R.sub.14 are each independently hydrogen, --CN, or selected from
the group consisting of alkyl, alkynyl, heteroalkyl, cycloalkyl,
heterocycloalkyl, aryl and heteroaryl, any of which is optionally
substituted with one or more fluorine atoms; [0355] Q.sub.1 and
Q.sub.2 are each independently absent or selected from a residue
which is hydrophilic at normal physiological pH; a conjugatable or
functionalizable residue; a residue which is hydrophobic at normal
physiological pH; an alkyl group optionally substituted with one or
more fluorine atoms; and a branched alkyl group optionally
substituted with one or more fluorine atoms; [0356] Q.sub.3 is a
residue which is positively charged at normal physiological pH;
[0357] Q.sub.4 is a residue which is negatively charged at normal
physiological pH, but undergoes protonation at lower pH; [0358] m
is a mole fraction of greater than 0.5 to less than 1.0; [0359] n
is a mole fraction of greater than 0 to less than 0.5; [0360] p is
a mole fraction of 0 to less than 0.5; wherein m+n+p=1; [0361] q is
a mole fraction of 0.1 to 0.9; [0362] r is a mole fraction of 0.05
to 0.9; [0363] s is present up to a mole fraction of 0.85; wherein
q+r+s=1; [0364] v is from 1 to 25 kDa; [0365] w is from 1 to 50
kDa; and [0366] at least one of Y.sub.1 and Q.sub.1 contains the
alkyl or branched alkyl group substituted with the one or more
fluorine atoms.
[0367] In some embodiments of a pH-sensitive polymer comprising a
copolymer of formula Ia as above, p is 0.
[0368] Suitable polymers of formula Ia include polymers where
R.sub.2-A.sub.1-Y.sub.1-Q.sub.1 taken together is a methacrylate
residue selected from the group consisting of
2,2,3,3,4,4,4-heptafluorobutyl methacrylate residue;
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl
2-methylacrylate residue; 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue; 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl
methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue; and
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue.
[0369] In various embodiments of a pH-sensitive polymer comprising
a copolymer of formula Ia as above, (a) Y.sub.3 is
--C(O)OCH.sub.2CH.sub.2, Q.sub.3 is dimethylamino, and/or R.sub.4
is --CH.sub.3; (b) Y.sub.4 is a covalent bond, Q.sub.4 is a
carboxyl residue, and/or R.sub.5 is --CH.sub.2CH.sub.2CH.sub.3; (c)
Y.sub.5 is --C(O)O(CH.sub.2).sub.3CH.sub.3 and/or R.sub.6 is
--CH.sub.3; and/or (d) Y.sub.0 is --C(O)O(2C-10C)alkyl-, Q.sub.0 is
O--[(C).sub.2-3--O].sub.x--R.sub.7 (where x is 1-48 and R.sub.7 is
--CH.sub.3), and/or R.sub.1 is --CH.sub.3. For example, in more
specific variations, R.sub.4-A.sub.3-Y.sub.3-Q.sub.3 taken together
is a dimethylaminoethyl methacrylate residue (DMAEMA);
R.sub.5-A.sub.4-Y.sub.4-Q.sub.4 taken together is a propyl acrylic
acid residue (PAA); R.sub.6-A.sub.5-Y.sub.5 taken together is a
butyl methacrylate residue (BMA); and/or
R.sub.1-A.sub.0-Y.sub.0-Q.sub.0 taken together is a
polyethyleneglycol methacrylate residue with 2-20 ethylene glycol
units (PEGMA).
[0370] In some embodiments of a polymer comprising a copolymer of
formula Ia as above, the pH-sensitive polymer is a polymer of
formula Va:
T1-L-[PEGMA.sub.m-M2n]v-[DMAEMA.sub.q-PAA.sub.r-BMA.sub.s].sub.w Va
[0371] where [0372] PEGMA is polyethyleneglycol methacrylate
residue with 2-20 ethylene glycol units; [0373] M2 is a
methacrylate residue selected from the group consisting of [0374] a
(C4-C18)alkyl-methacrylate residue substituted with one or more
fluorine atoms, and [0375] a (C4-C18)branched alkyl-methacrylate
residue substituted with one or more fluorine atoms, [0376] BMA is
butyl methacrylate residue; [0377] PAA is propyl acrylic acid
residue; [0378] DMAEMA is dimethylaminoethyl methacrylate residue;
[0379] m and n are each a mole fraction greater than 0, where m is
greater than n and m+n=1; [0380] q is a mole fraction of 0.2 to
0.75; [0381] r is a mole fraction of 0.05 to 0.6; [0382] s is a
mole fraction of 0.2 to 0.75; [0383] q+r+s=1; [0384] v is 1 to 25
kDa; [0385] w is 1 to 25 kDa; [0386] T1 is absent or is the first
targeting ligand; and [0387] L is absent or is a linking
moiety.
[0388] Particularly suitable M2 methacrylate residues for use in a
polymer of formula Va include 2,2,3,3,4,4,4-heptafluorobutyl
methacrylate residue;
3,3,4,4,5,6,6,6-octafluoro-5(trifluoromethyl)hexyl methacrylate
residue; 2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-pentadecafluorooctyl
2-methylacrylate residue 3,3,4,4,5,5,6,6,6-nonafluorohexyl
methacrylate residue; 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl
methacrylate residue;
1,1,1-trifluoro-2-(trifluoromethyl)-2-hydroxy-4-methyl-5-pentyl
methacrylate residue; and
2-[(1',1',1'-trifluoro-2'-(trifluoromethyl)-2'-hydroxy)propyl]-3-norborny-
l methacrylate residue.
[0389] In particular variations of a polymer of formula V, formula
Va, or any of formulae Vb-Vm, m is from 0.55 to 0.9 (e.g., from
0.65 to 0.9 or from 0.7 to 0.85), n is from 0.1 to 0.45 (e.g., from
0.1 to 0.35 or from 0.15 to 0.3), q is from 0.25 to 0.4 (e.g., 0.28
to 0.37), r is from 0.07 to 0.15 (e.g., 0.9 to 0.15), s is from 0.5
to 0.65 (e.g., 0.5 to 0.6), v is from 2.5 kDa to 10 kDa (e.g., from
2.5 kDa to 7 kDa, from 2.5 kDa to 5 kDa, from 2.5 kDa to 4.5 kDa,
or from 0.29 to 4 kDa), and/or w is from 4 kDa to 9 kDa (e.g., from
4 kDa to 7 kDa, from 4 kDa to 6 kDa, or from 5 kDa to 7 kDa). In
some specific embodiments, the ratio of w:v ranges from about 1:0.8
to about 5:1, or from about 1:1 to about 2:1.
[0390] Generally, a membrane-destabilizing polymer (or polymer
chains included as constituent moieties such as blocks of a block
copolymer) can include a shielding agent or solubilizing agent. The
shielding agent can be effective for improving solubility of the
polymer chain. The shielding agent can also be effective for
reducing toxicity of the certain compositions. In some embodiments,
the shielding agent can be a polymer comprising a plurality of
neutral hydrophilic monomeric residues. The shielding polymer can
be covalently coupled to a membrane destabilizing polymer, directly
or indirectly, through an end group of the polymer or through a
pendant functional group of one or more monomeric residues of the
polymer. In some embodiments, a plurality of monomeric residues of
the polymer chain can have a shielding species; preferably, such
shielding species is a pendant moiety from a polymerizable monomer
(from which the shielding monomeric residues are derived). For
example, the polymer can comprise a plurality of monomeric residues
having a pendant group comprising a shielding oligomer. A
shielding/solubilizing species may be conjugated to a polymer via a
labile linkage such as, for example, a pH-sensitive bond or linker.
Particularly suitable pH-sensitive bonds and linkers include
hydrazone, acetal, ketal, imine, orthoester, carbonate, and
maleamic acid linkages. Labile linkages may be utilized, e.g., for
linkage via a plurality of monomeric residues having pendant
linking groups or for linkage of a polymer block comprising the
shielding species to another polymer block (e.g., linkage of a
shielding block to a membrane-destabilizing block).
[0391] A preferred shielding/solubilizing polymer can be a
polyethylene glycol (PEG) oligomer (e.g., having 20 or less repeat
units) or polymer (e.g., having more than 20 repeat units). PEG can
be described as a polyethylene glycol or as a polyethylene oxide,
and is understood to be a oligomer or polymer from --CH2--CH2-O--
repeat units (which repeat units are also referred to herein as
"ethylene glycol units" or "ethylene oxide units"). In certain
embodiments, one block of a block copolymer can be or comprises a
polyethylene glycol (PEG) oligomer or polymer--for example,
covalently coupled to the alpha end or the omega end of the
membrane destabilizing block of the copolymer. In another
embodiment, a polyethylene glycol (PEG) oligomer or polymer can be
covalently coupled to the polymer through a conjugating monomeric
residue having a species which includes a functional group suitable
for linking, directly or indirectly, to the polyethylene glycol
oligomer or polymer. In another embodiment, the monomeric residue
can be derived from a polymerizable monomer which includes a
polyethylene glycol oligomer pendant to the monomer (e.g., PEGMA as
described above).
[0392] In one general approach, PEG chains or blocks are covalently
coupled to a membrane-destabilizing polymer chain. For such
embodiments, for example, PEG chains or blocks can have molecular
weights ranging approximately from 1,000 to approximately 30,000.
In some embodiments, the PEG is effective as (i.e., is incorporated
into) a second block of a block copolymer. For example, PEG can be
a second block coupled covalently to a block comprising a membrane
destabilizing polymer. In some embodiments, PEG is conjugated to
block copolymer ends groups, or to one or more pendant modifiable
group present in polymeric compound, such as conjugated to
modifiable groups within a hydrophilic segment or block (e.g., a
second block) of a polymer (e.g., block copolymer). As an example,
a block of a copolymer can be or can be conjugated to a shielding
polymer having a repeat unit of formula III
##STR00005##
[0393] where R.sup.1 and R.sup.2 are each independently selected
from the group consisting of hydrogen, halogen, hydroxyl, and
optionally substituted C.sub.1-C.sub.3 alkyl, and having a
molecular weight ranging from about 1,500 to about 15,000.
[0394] In another general approach, a monomeric residue is derived
from a polymerizable monomer comprising a PEG oligomer; for
example, such monomeric residues can be incorporated into the
polymer or into one or more blocks of a block copolymer during
polymerization. In preferred embodiments, monomeric residues can be
derived from a polymerizable monomer having a pendant group
comprising an oligomer of formula IV
##STR00006##
[0395] where R.sup.1 and R.sup.2 are each independently selected
from the group consisting of hydrogen, halogen, hydroxyl, and
optionally substituted C.sub.1-C.sub.3 alkyl, and n is an integer
ranging from 2 to 20.
[0396] Generally, a membrane-destabilizing polymer (or polymer
chains included as constituent moieties such as blocks of a block
copolymer) can be prepared in any suitable manner. Suitable
synthetic methods used to produce, for example, a
membrane-destabilizing copolymer include, by way of non-limiting
example, well-known "living polymerization" methods such as, e.g.,
cationic, anionic and free radical polymerization.
[0397] Using living polymerization, polymers of very low
polydispersity or differences in chain length can be obtained.
Polydispersity is usually measured by dividing the weight average
molecular weight of the polymer chains by their number average
molecular weight. The number average molecule weight is sum of
individual chain molecular weights divided by the number of chains.
The weight average molecular weight is proportional to the square
of the molecular weight divided by the number of molecules of that
molecular weight. Since the weight average molecular weight is
always greater than the number average molecular weight,
polydispersity is always greater than or equal to one. As the
numbers come closer and closer to being the same, i.e., as the
polydispersity approaches a value of one, the polymer becomes
closer to being monodisperse in which every chain has exactly the
same number of constitutional units. Polydispersity values
approaching one are achievable using radical living polymerization.
Methods of determining polydispersity such as, without limitation,
size exclusion chromatography, dynamic light scattering,
matrix-assisted laser desorption/ionization mass spectrometry, and
electrospray mass spectrometry are well-known in the art.
[0398] Reversible addition-fragmentation chain transfer or RAFT is
an exemplary living polymerization technique for use in
synthesizing ethylenic backbone polymers. RAFT is well-known to
those skilled in the art. RAFT comprises a free radical
degenerative chain transfer process. Most RAFT procedures employ
thiocarbonylthio compounds such as, without limitation,
dithioesters, dithiocarbamates, trithiocarbonates and xanthates to
mediate polymerization by a reversible chain transfer mechanism.
Reaction of a polymeric radical with the C.dbd.S group of any of
the preceding compounds leads to the formation of stabilized
radical intermediates. These stabilized radical intermediates do
not undergo the termination reactions typical of standard radical
polymerization but, rather, reintroduce a radical capable of
re-initiation or propagation with monomer, reforming the C.dbd.S
bond in the process. This cycle of addition to the C.dbd.S bond
followed by fragmentation of the ensuing radical continues until
all monomer has been consumed or the reaction is quenched. The low
concentration of active radicals at any particular time limits
normal termination reactions. In other embodiments, polymers are
synthesized by Macromolecular design via reversible
addition-fragmentation chain transfer of Xanthates (MADIX) (Direct
Synthesis of Double Hydrophilic Statistical Di- and Triblock
Copolymers Comprised of Acrylamide and Acrylic Acid Units via the
MADIX Process", Daniel Taton et al., Macromolecular Rapid
Communications, 22:1497-1503, 2001.)
[0399] In certain embodiments of the present invention, the lipid
nanoparticle and/or the membrane destabilizing polymer includes at
least one targeting ligand that specifically binds to a molecule on
the surface of the target cell. In some embodiments, the
membrane-destabilizing polymer comprises the targeting ligand. In
some embodiments, the lipid nanoparticle comprises the targeting
ligand. In some embodiments, both the membrane-destabilizing
polymer and the lipid nanoparticle comprise a target ligand, which
may be the same or different (e.g., different targeting ligand
species that bind to the same target cell).
[0400] A targeting ligand specifically recognizes a molecule on the
surface of the target cell, such as, e.g., a cell surface receptor.
Particularly suitable targeting moieties include antibodies,
antibody-like molecules, polypeptides, proteins (e.g., insulin-like
growth factor II (IGF-II)), peptides (e.g., an integrin-binding
peptide such as an RGD-containing peptide), and small molecules
such as, for example, sugars (e.g., lactose, galactose, N-acetyl
galactosamine (NAG), mannose, mannose-6-phosphate (M6P)) or
vitamins (e.g., folate). In some variations, a targeting moiety is
a protein derived from a natural ligand of a cell-surface molecule
(e.g., derived from a cytokine or from the extracellular domain of
a cell-surface receptor that binds to a cell surface
counter-receptor). Examples of cell surface molecules that may be
targeted by a targeting moiety of a copolymer provided herein
include, but are not limited to, the transferrin receptor type 1
and 2, the EGF receptor, HER2/Neu, VEGF receptors, integrins, NGF,
CD2, CD3, CD4, CD8, CD19, CD20, CD22, CD33, CD43, CD38, CD56, CD69,
the asialoglycoprotein receptor, mannose receptor, the
cation-independent mannose-6-phosphate/IGF-II receptor,
prostate-specific membrane antigen (PSMA), a folate receptor, and a
sigma receptor.
[0401] In particular variations, a targeting ligand includes an
N-acetylgalactosamine (NAG) sugar residue, which specifically binds
to the asialoglycoprotein receptor (ASGPR) on hepatocytes. In some
such embodiments, the targeting ligand has the formula
##STR00007##
In other embodiments comprising a NAG sugar residue, the targeting
ligand comprises multiple NAG sugar residues (e.g., three NAG
residues, also referred to herein as a "tri-NAG" structure), which
may increase avidity for the asialoglycoprotein receptor relative
to a monovalent NAG moiety. In some such embodiments, a tri-NAG
structure has the formula
##STR00008##
where designates a point of attachment.
[0402] In various embodiments, a targeting ligand is attached to
either end of a membrane-destabilizing polymer (e.g., block
copolymer), attached to a side chain of a monomeric unit,
incorporated into a polymer block, or attached to a lipid or
polymeric component of a lipid nanoparticle. Attachment of a
targeting ligand to the membrane-destabilizing polymer or LNP is
achieved in any suitable manner, e.g., by any one of a number of
conjugation chemistry approaches including, but not limited to,
amine-carboxyl linkers, amine-sulfhydryl linkers,
amine-carbohydrate linkers, amine-hydroxyl linkers, amine-amine
linkers, carboxyl-sulfhydryl linkers, carboxyl-carbohydrate
linkers, carboxyl-hydroxyl linkers, carboxyl-carboxyl linkers,
sulfhydryl-carbohydrate linkers, sulfhydryl-hydroxyl linkers,
sulfhydryl-sulfhydryl linkers, carbohydrate-hydroxyl linkers,
carbohydrate-carbohydrate linkers, and hydroxyl-hydroxyl linkers.
In specific embodiments, "click" chemistry is used to attach the
targeting ligand to a polymer (for example of "click" reactions,
see Wu and Fokin, "Catalytic Azide-Alkyne Cycloaddition: Reactivity
and Applications," Aldrichim. Acta 40:7-17, 2007). A large variety
of conjugation chemistries are optionally utilized (see, e.g.,
Bioconjugation, Aslam and Dent, Eds, Macmillan, 1998 and chapters
therein). In some embodiments, targeting ligands are attached to a
monomer and the resulting compound is then used in the
polymerization synthesis of a polymer (e.g., block copolymer). In
some embodiments, targeting moieties are attached to a block of a
first block copolymer, or to a block of a second block copolymer in
a mixed polymer micellic assembly.
[0403] Targeting of lipid particles using a variety of targeting
ligands has been previously described. See, e.g., U.S. Pat. Nos.
4,957,773 and 4,603,044. Targeting mechanisms generally require
that the targeting ligand be positioned on the surface of the lipid
particle in such a manner that the targeting moiety is available
for interaction with the target, for example, a cell surface
receptor. A variety of different targeting ligands and methods are
known and available in the art, including those described above as
well as, e.g., in Sapra and Allen, Prog. Lipid Res. 42:439-62,
2003, and Abra et al., J. Liposome Res. 12:1-3, 2002. Various
targeting counter-receptors can be bound to the surface of the
liposome, such as antibodies, antibody fragments, carbohydrates,
vitamins, and transport proteins. For example, for targeting to the
liver, liposomes can be modified with branched type galactosyllipid
derivatives to target asialoglycoprotein receptors. See Kato and
Sugiyama, Crit. Rev. Ther. Drug Carrier Syst. 14:287, 1997;
Murahashi et al., Biol. Pharm. Bull. 20:259, 1997. In a more
general approach to tissue targeting, target cells are prelabeled
with biotinylated antibodies specific for a molecule expressed by
the target cell. See Harasym et al., Adv. Drug Deliv. Rev. 32:99,
1998. After plasma elimination of free antibody,
streptavidin-conjugated liposomes are administered. In another
approach, targeting antibodies are directly attached to liposomes.
See Harasym et al., supra.
[0404] In specific variations, a targeting ligand is attached to a
polymer using a linker having a formula selected from
##STR00009## ##STR00010##
where m is 1-100 or 10-250 and each of w, x, y, and z is
independently 1-48. In certain variations of a linker comprising m
as above, m is 1-15, 10-20, 20-30, 20-25, 11 or 12. In other
variations of a linker comprising m as above, m is 20-60, 25-60,
25-55, 25-50, 25-48, 30-60, 30-55, 30-50, 30-48, 34-60, 34-55,
34-50, 34-48, 36-60, 36-55, 36-50, 36-48, 36, or 48. In yet other
embodiments of a linker comprising m as above, m is 60-250,
100-250, 150-250, or 200-250. In certain variations of L1
comprising x and y, x, y and z, or w, x, y and z as above, each of
w, x, y, and z is independently 20-30, 20-25, or 23. In other
variations of L1 comprising x and y, x, y and z, or w, x, y and z
as above, each of w, x, y, and z is independently 1-12, 1-24, 1-36,
8-16, 10-14, 20-28, 22-26, 32-40, 34-38, 8-48, 10-48, 20-48, 22-48,
32-48, 34-48, or 44-48.
[0405] Particular embodiments of the present invention are directed
at in vivo delivery of therapeutic agents. In some embodiments, the
therapeutic agent is a polynucleotide. Suitable polynucleotide
therapeutic agents include DNA agents, which may be in the form of
cDNA, in vitro polymerized DNA, plasmid DNA, genetic material
derived from a virus, linear DNA, vectors, expression vectors,
expression cassettes, chimeric sequences, recombinant DNA,
anti-sense DNA, or derivatives of these groups. Other suitable
polynucleotide therapeutic agents include RNA, which may be in the
form of messenger RNA (mRNA), in vitro polymerized RNA, recombinant
RNA, transfer RNA (tRNA), small nuclear RNA (snRNA), ribosomal RNA
(rRNA), chimeric sequences, dicer substrate and the precursors
thereof, locked nucleic acids, anti-sense RNA, interfering RNA
(RNAi), asymmetric interfering RNA (aiRNA), small interfering RNA
(siRNA), microRNA (miRNA), ribozymes, external guide sequences,
small non-messenger RNAs (snmRNA), untranslatedRNA (utRNA), snoRNAs
(24-mers, modified snmRNA that act by an anti-sense mechanism),
tiny non-coding RNAs (tncRNAs), small hairpin RNA (shRNA), or their
derivatives. Double stranded RNA (dsRNA) and siRNA are of interest
particularly in connection with the phenomenon of RNA interference.
Examples of therapeutic oligonucleotides as used herein include,
but are not limited to, siRNA, an antisense oligonucleotide, a
dicer substrate, a miRNA, an aiRNA or an shRNA. An example of a
large therapeutic polynucleotide as used herein includes, but is
not limited to, messenger RNAs (mRNAs) encoding functional proteins
for gene replacement therapy. Polynucleotide therapeutic agents may
also be nucleic acid aptamers, which are nucleic acid oligomers
that specifically bind other macromolecules; such aptamers that
bind specifically to other macromolecules can be readily isolated
from libraries of such oligomers by known technologies such as
SELEX. See, e.g., Stoltenburg et al., Biomol. Eng., 24:381,
2007.
[0406] In other embodiments, the therapeutic agent is a protein or
a peptide. For example, in certain variations, the agent is an
antibody that binds to and either antagonizes or agonizes an
intracellular target. Antibodies for use in the present invention
may be raised through any known method, such as through injection
of immunogen into mice and subsequent fusions of lymphocytes to
create hybridomas. Such hybridomas may then be used either (a) to
produce antibody directly, or (b) to clone cDNAs encoding antibody
fragments for subsequent genetic manipulation. To illustrate one
method employing the latter strategy, mRNA is isolated from the
hybridoma cells, reverse-transcribed into cDNA using antisense
oligo-dT or immunoglobulin gene-specific primers, and cloned into a
plasmid vector. Clones are sequenced and characterized. They may
then be engineered according to standard protocols to combine the
heavy and light chains of the antibody into a bacterial or
mammalian expression vector to generate, e.g., a single-chain scFv.
A similar approach may be used to generate recombinant bispecific
antibodies by combining the heavy and light chains of each of two
different antibodies, separated by a short peptide linker, into a
bacterial or mammalian expression vector. Recombinant antibodies
are then expressed and purified according to well-established
protocols in bacteria or mammalian cells. See, e.g., Kufer et al.,
2004, supra; Antibody Engineering: A Practical Approach,
McCafferty, Hoogenboom and Chiswell Eds, IRL Press 1996. Antibodies
or other proteinaceous therapeutic molecules such as peptides, may
also be created through display technologies that allow selection
of interacting affinity reagents through the screening of very
large libraries of, for example, immunoglobulin domains or peptides
expressed by bacteriophage (Antibody Engineering: A Practical
Approach, McCafferty, Hoogenboom and Chiswell Eds, IRL Press 1996).
Antibodies may also be humanized through grafting of human
immunoglobulin domains, or made from transgenic mice or
bacteriophage libraries that have human immunoglobulin genes/cDNAs.
In some embodiments of the invention, a specific binding protein
therapeutic may include structures other than antibodies that are
able to bind to targets specifically, including but not limited to
avimers (see Silverman et al., Nature Biotechnology 23:1556-1561,
2005), ankyrin repeats (see Zahnd et al., J. Mol. Biol.
369:1015-1028, 2007) and adnectins (see U.S. Pat. No. 7,115,396),
and other such proteins with domains that can be evolved to
generate specific affinity for antigens, collectively referred to
as "antibody-like molecules". Modifications of protein therapeutics
through the incorporation of unnatural amino acids during synthesis
may be used to improve their properties (see Datta et al., J. Am.
Chem. Soc. 124:5652-5653, 2002; and Liu et al., Nat. Methods
4:239-244, 2007). Such modifications may have several benefits,
including the addition of chemical groups that facilitate
subsequent conjugation reactions.
[0407] In some embodiments, the therapeutic agent is a peptide. In
certain variations, the peptide is a bispecific peptide. Peptides
can readily be made and screened to create affinity reagents that
recognize and bind to macromolecules such as, e.g., proteins. See,
e.g., Johnsson and Ge, Current Topics in Microbiology and
Immunology, 243:87-105, 1999.
[0408] In other embodiments, a protein therapeutic is a peptide
aptamer. A peptide aptamer comprises a peptide molecule that
specifically binds to a target protein and interferes with the
functional ability of that target protein. See, e.g., Kolonin et
al., Proc. Natl. Acad. Sci. USA 95:14266, 1998. Peptide aptamers
consist of a variable peptide loop attached at both ends of a
protein scaffold. Such peptide aptamers can often have a binding
affinity comparable to that of an antibody (nanomolar range). Due
to the highly selective nature of peptide aptamers, they can be
used not only to target a specific protein, but also to target
specific functions of a given protein (e.g., a signaling function).
Further, peptide aptamers can be expressed in a controlled fashion
by use of promoters that regulate expression in a temporal, spatial
or inducible manner. Peptide aptamers act dominantly, therefore,
they can be used to analyze proteins for which loss-of-function
mutants are not available. Peptide aptamers are usually prepared by
selecting the aptamer for its binding affinity with the specific
target from a random pool or library of peptides. Peptide aptamers
can be isolated from random peptide libraries by yeast two-hybrid
screens. See, e.g., Xu et al., Proc. Natl. Acad. Sci. USA 94:12473,
1997. They can also be isolated from phage libraries (see, e.g.,
Hoogenboom et al., Immunotechnology 4:1, 1998) or from chemically
generated peptides/libraries.
[0409] In yet other embodiments, the therapeutic agent is a small
molecule therapeutic. Small molecule therapeutics are generally
well-known in the art and may be used in accordance with the
present invention. Such molecules include anti-infective (e.g.,
anti-viral) small molecules, immunomodulatory small molecules, and
anti-cancer small molecules, to name a few broad categories. In
some variations, the small molecule therapeutic is a hydrophobic
small molecule. Small molecule anti-cancer therapeutics include,
e.g., a variety of chemotherapeutic drugs such as, for example,
tyrosine kinase inhibitors (TKIs), small molecules that influence
either DNA or RNA, or small molecules that inhibit cell mitosis by
preventing polymerization or depolymerization of microtubules.
Particular examples of small molecule chemotherapeutic agents
include anti-metabolites (such as Azathioprine, Cytarabine,
Fludarabine phosphate, Fludarabine, Gemcitabine, cytarabine,
Cladribine, capecitabine 6-mercaptopurine, 6-thioguanine,
methotrexate, 5-fluoroouracil and hyroxyurea); alkylating agents
(such as Melphalan, Busulfan, Cis-platin, Carboplatin,
Cyclophosphamide, Ifosphamide, Dacarabazine, Procarbazine,
Chlorambucil, Thiotepa, Lomustine, Temozolamide); anti-mitotic
agents (such as Vinorelbine, Vincristine, Vinblastine, Docetaxel,
Paclitaxel); topoisomerase inhibitors (such as Doxorubincin,
Amsacrine, Irinotecan, Daunorubicin, Epirubicin, Mitomycin,
Mitoxantrone, Idarubicin, Teniposide, Etoposide, Topotecan);
antibiotics (such as Actinomycin and Bleomycin); Asparaginase;
anthracyclines; and taxanes. In certain variations, the small
molecule chemotherapeutic is selected from an anti-tubulin agent, a
DNA minor groove binding agent, a DNA replication inhibitor, and a
tyrosine kinase inhibitor. In other specific variations, the small
molecule chemotherapeutic is an anthracycline, an auristatin, a
camptothecin, a duocarmycin, an etoposide, a maytansinoid, a vinca
alkaloid, or a platinum (II) compound.
[0410] In still other embodiments, the therapeutic agent is a
component of a gene editing system that disrupts or corrects genes
that cause disease. These include, for example, zinc finger
nucleases (ZFNs) (see, e.g., Smith et al., Nucleic Acids Res.
28:3361-3369, 2000), transcription activator-like effector
nucleases (TALENs) (see, e.g., Li et al., Nucleic Acids Res.
39:359-372, 2011), the CRISPR/Cas system (see, e.g., Richter et
al., Int. J. Mol. Sci. 14:14518-14531, 2013), and engineered
meganucleases (see, e.g., Silva et al., Curr. Gene Ther. 11:11-27,
2011). In such embodiments, the nuclease(s) are encoded by one or
more nucleic acids such as mRNA or DNA that are formulated in the
lipid nanoparticle. In some variations, multiple mRNAs are
formulated in the LNP carrier to deliver two nucleases to the same
cell for gene editing to occur (e.g., for a ZFNs or TALENs gene
editing system, which typically requires two nucleases to recognize
the specific target site within the genome to cause a modification
at that site). In the context of the present disclosure, the
membrane destabilizing polymer facilitates delivery of the nucleic
acid(s) to the cytoplasm, where translation or subsequent nuclear
delivery occur. In some variations, one or more additional
components of a gene editing system are delivered to a target cell
together with the one or more nucleic acids encoding the
nuclease(s). For example, in the CRISPR/Cas system, in addition to
a nucleic acid encoding the Cas9 protein, a short guide RNA to
target the enzyme to a specific site in the genome is typically
formulated within the LNP carrier. In certain embodiments, to
correct a gene by homologous recombination, a donor DNA sequence
may also be delivered and formulated either in the same or a
different LNP than with the nucleic acid(s) that encode the
nuclease(s). In certain embodiments where the gene editing system
corrects a gene associated with a disease, the disease is
characterized by deficiency of a functional protein as disclosed
herein (see, e.g., discussion of protein deficiency diseases,
infra.)
[0411] In some embodiments, the therapeutic agent is an immunogen.
Using methods as disclosed herein, an immunogen can be effectively
delivered to a variety of immune cells to elicit an immune
response. In some variations, only the LNP comprises an immunogen.
In other embodiments, the membrane destabilizing polymer is also
associated with (e.g., covalently coupled to) an immunogen.
Suitable immunogens include peptides, proteins, mRNAs, short RNAs,
DNAs, simple or complex carbohydrates as well as substances derived
from viruses, bacteria, cancer cells, and the like. In some
variations, a hapten or adjuvant component is attached (conjugated)
or self-associated with the membrane destabilizing polymer or the
LNP. In certain embodiments in which both the membrane
destabilizing polymer and LNP are associated with an immunogen, the
immunogen associated with the polymer is different than that for
the LNP; alternatively, both the polymer and LNP have the same
immunogenic cargo. For example, in some variations, a immunogenic
peptide that is a promiscuous T-cell epitope is attached to the
membrane destabilizing polymer or the LNP to enable a more robust
immune response. This hapten can be derived from, e.g., the protein
sequence encoded by an mRNA component of the LNP or can be from
another protein or a combination of more than one T-cell epitope.
As another example, the immunogen may be a component of a bacterial
cell wall that is attached to the polymer or LNP to enhance the
immune response by acting as an adjuvant. In yet other variations,
an immmunostimulating oligonucleotide or long nucleic acid is
attached or self-associated with the polymer or LNP to activate the
innate immune response. Utilizing the dual nature of the delivery
system described herein (using both a membrane destabilizing
polymer component and an LNP component), one component may be used
to initiate a T-cell response while the other component is utilized
to initiate a B-cell response. The polymer and LNP components of
the hybrid delivery system may be used to elicit an innate immune
response, a T-cell response, a B-cell response, or a combination
thereof through the attachment or self-association of immunogenic
substances. In some embodiments, a first polymer is used to attach
and carry an immunogen while a second, membrane destabilizing
polymer is used to enable uptake into antigen presenting cells. In
certain embodiments for delivering an immunogen to a cell as
disclosed herein, at least one of the polymer and the LNP has a
targeting ligand to direct the polymer and/or LNP to an immune cell
of interest.
[0412] For delivery of a therapeutic or diagnostic agent to the
cytosol of a target cell (e.g., for delivery to a target tissue
comprising the target cells), a membrane-destabilizing polymer and
a lipid nanoparticle comprising the therapeutic or diagnostic agent
are each administered to a subject in amounts effective to achieve
intracellular delivery of the agent. The lipid nanoparticle and
membrane-destabilizing polymer may be co-formulated as a single
composition for co-injection into a subject. Alternatively, the
lipid nanoparticle and membrane-destabilizing polymer may be
formulated separately for separate administration. Typically, for
separate administration, the lipid nanoparticle and
membrane-destabilizing polymer are administered sequentially. For
example, in particular embodiments, the membrane-destabilizing
polymer is administered after administration of the lipid
nanoparticle. In specific variations, the timing between
administration of LNP and polymer is about two hours or less,
typically about one hour or less, and more typically about 30
minutes or less, about 10 minutes or less, about five minutes or
less, or about one minute or less. In some embodiments, the timing
between administration of LNP and polymer is about 30 minutes,
about 15 minutes, about 10 minutes, about five minutes, or about
one minute. Typically, in variations comprising co-injection of the
lipid nanoparticle and membrane-destabilizing polymer, the LNP and
polymer are initially formulated as separate compositions and then
mixed together into a single composition prior to
administration.
[0413] Any cell type or corresponding tissue may be targeted for
agent delivery using the present methods. Suitable target cells
include, e.g., chondrocytes, epithelial cells, nerve cells, muscle
cells, blood cells (e.g., lymphocytes or myeloid leukocytes),
endothelial cells, pericytes, fibroblasts, glial cells, and
dendritic cells. Other suitable target cells include cancer cells,
immune cells, bacterially-infected cells, virally-infected cells,
or cells having an abnormal metabolic activity. In a particular
variation where the target cell is a secretory cell, the target
secretory cell is a hepatocyte. In some such embodiments, either or
both of the LNP and membrane-destabilizing polymer includes a
targeting ligand that specifically binds to the asialoglycoprotein
receptor (ASGPR); for example, in particular variations, a
targeting ligand includes an N-acetylgalactosamine (NAG) residue
(e.g., a monovalent NAG moiety or a tri-NAG structure). Target
cells further include those where the cell is in a mammalian
animal, including, for example, a human, rodent, murine, bovine,
canine, feline, sheep, equine, and simian mammal.
[0414] In particular embodiments comprising delivery of a
polynucleotide, the polynucleotide is an mRNA molecule encoding a
functional protein, such as a functional protein associated with a
protein deficiency disease, and the method increases the amount of
the functional protein within the target cell. For example, in
specific variations, the mRNA encodes a protein selected from
erythropoietin, thrombopoietin, Factor VII, Factor VIII, LDL
receptor, alpha-1-antitrypsin (A1AT), carbamoyl phosphate
synthetase I (CPS1), fumarylacetoacetase (FAH) enzyme,
alanine:glyoxylate-aminotransferase (AGT), methylmalonyl CoA mutase
(MUT), propionyl CoA carboxylase alpha subunit (PCCA), propionyl
CoA carboxylase beta subunit (PCCB), a subunit of branched-chain
ketoacid dehydrogenase (BCKDH), ornithine transcarbamylase (OTC),
copper-transporting ATPase Atp7B, bilirubin uridinediphosphate
glucuronyltransferase (BGT) enzyme, hepcidin, glucose-6-phosphatase
(G6Pase), glucose 6-phosphate translocase, lysosomal
glucocerebrosidase (GB), Niemann-Pick C1 protein (NPC1),
Niemann-Pick C2 protein (NPC2), acid sphingomyelinase (ASM), Factor
IX, galactose-1-phosphate uridylyltransferase, galactokinase,
UDP-galactose 4-epimerase, transthyretin, a complement regulatory
protein, phenylalanine hydroxylase (PAH), homogentisate
1,2-dioxygenase, porphobilinogen deaminase, hypoxanthine-guanine
phosphoribosyltransferase (HGPRT), argininosuccinate lyase (ASL),
argininosuccinate synthetase (ASS1), P-type ATPase protein FIC-1,
alpha-galactosidase A, acid ceramidase, acid .alpha.-L-fucosidase,
acid f-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase,
galactocerebrosidase, acid .alpha.-mannosidase, .beta.-mannosidase,
arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate
sulfatase, acid f-galactosidase, acid .alpha.-glucosidase,
.beta.-hexosaminidase B, heparan-N-sulfatase,
alpha-N-acetylglucosaminidase, acetyl-CoA:.alpha.-glucosaminide
N-acetyltransferase, N-acetylglucosamine-6-sulfate sulfatase,
alpha-N-acetylgalactosaminidase, sialidase, .beta.-glucuronidase,
and .beta.-hexosaminidase A.
[0415] In certain embodiments comprising delivery of an mRNA
molecule encoding a functional protein, the mRNA encodes a secreted
protein. Exemplary secreted proteins include erythropoietin,
thrombopoietin, granulocyte-colony stimulating factor, granulocyte
macrophage-colony stimulating factor, leptin, platelet-derived
growth factors (e.g., platelet-derived growth factor B),
keratinocyte growth factor, bone morphogenic protein 2, bone
morphogenic protein 7, insulin, glucagon-like peptide-1, human
growth hormone, clotting factors (e.g., Factor VII, Factor VIII,
Factor IX), relaxins (e.g., relaxin-2), interferons (e.g.,
interferon-.alpha., interferon-.beta., interferon-.gamma.),
interleukins (e.g., interleukin-2, interleukin-4, interleukin-10,
interleukin-11, interleukin-12, interleukin-18, interleukin-21),
and chemokines (e.g., CC subfamily chemokines, CXC subfamily
chemokines, C subfamily chemokines, CX3C subfamily chemokines).
Secreted proteins also include antibodies, which may be selected
from various antibody embodiments described herein. Particularly
suitable antibodies include genetically engineered antibodies such
as, for example, chimeric antibodies, humanized antibodies,
single-chain antibodies (e.g., a single-chain Fv (scFv)), and
bispecific antibodies. In some variations, the mRNA encodes an
antibody that specifically binds and antagonizes a protein selected
from vascular endothelial growth factor A (VEGF-A), tumor necrosis
factor .alpha. (TNF.alpha.), interleukin-6 (IL-6), interleukin-17A
(IL-17A), interleukin-17F (IL-17F), interleukin-21 (IL-21),
interleukin-23 (IL-23), cytotoxic T-lymphocyte-associated protein 4
(CTLA-4), and programmed cell death protein 1 (PD-1).
[0416] In certain embodiments comprising increasing the amount of a
protein in a cell, the protein is ornithine transcarbamylase (OTC).
In such embodiments, an mRNA encoding an OTC protein is formulated
into a lipid nanoparticle composition and is administered to a
subject with co-injection or separate injection of a
membrane-destabilizing polymer as described herein. In particular
variations, the mRNA molecule encodes an OTC protein comprising an
amino acid sequence having at least 90% or at least 95% sequence
identity with residues 35-354 of SEQ ID NO:1 (e.g., at least 96%,
at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%
sequence identity with residues 35-354 of SEQ ID NO:1). To direct
an encoded OTC protein to the mitochondria of the cell, an mRNA
molecule encoding the OTC protein includes a sequence encoding a
mitochondrial targeting signal peptide (also referred to herein as
a "mitochondrial leader sequence"). The mitochondrial leader
sequence may be that of a native OTC protein (e.g., residues 1-34
of SEQ ID NO:1 (a native human mitochondrial leader sequence) or
residues 1-34 of SEQ ID NO:2 (a native mouse mitochondrial leader
sequence)), or may be derived from another protein comprising a
mitochondrial targeting signal peptide, or synthesized de novo. An
engineered cleavage site may be included at the junction between
the mitochondrial leader sequence and the remainder of the
polypeptide to optimize proteolytic processing in the cell. The
mitochondrial leader sequence is operably linked to the mRNA
sequence encoding the mature OTC protein, i.e., the two sequences
are joined in the correct reading frame and positioned to direct
the newly synthesized polypeptide to the mitochondria of a cell.
Mitochondrial leader sequences are commonly positioned at the amino
terminus of the protein. In specific variations, the encoded OTC
protein with a mitochondrial leader sequence has an amino acid
sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2. Suitable mRNA
sequences encoding an OTC protein of SEQ ID NO:1, and which may be
formulated into a lipid nanoparticle composition, may comprise
sequences as shown in SEQ ID NO:6 or SEQ ID NO:8 (coding sequence
(CDS) for each corresponding to residues 48-1112). Suitable mRNA
sequences encoding an OTC protein of SEQ ID NO:2, and which may be
formulated into a lipid nanoparticle composition, may comprise a
sequence as shown in SEQ ID NO:7 (coding sequence (CDS)
corresponding to residues 48-1112). An OTC-encoding mRNA for
formulation with a lipid nanoparticle typically further includes a
poly(A) at its 3' end (e.g., a polyA tail of from about 50 to about
500 adenine residues), which may be added to a construct using
well-known genetic engineering techniques (e.g., via PCR).
Exemplary DNA sequences that may be used for insertion into an
appropriate DNA vector for production and preparation of mRNA
constructs of SEQ ID NOs. 6-8 are shown in SEQ ID NOs. 3-5,
respectively.
[0417] In other embodiments comprising increasing the amount of a
protein in a cell, the protein is methylmalonyl CoA mutase (MUT),
propionyl CoA carboxylase subunit A (PCCA), propionyl CoA
carboxylase subunit B (PCCB), or a subunit of branched-chain
ketoacid dehydrogenase (BCKDH). In such embodiments, an mRNA
encoding a MUT, PCCA, PCCB, or BCKDH subunit protein is formulated
into a lipid nanoparticle composition and is administered to a
subject with co-injection or separate injection of a
membrane-destabilizing polymer as described herein. In particular
variations, the mRNA molecule encodes a MUT protein comprising an
amino acid sequence having at least 90% or at least 95% sequence
identity with residues 33-750 of SEQ ID NO:9 (e.g., at least 96%,
at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%
sequence identity with residues 33-750 of SEQ ID NO:9). In other
variations, the mRNA molecule encodes a PCCA protein comprising an
amino acid sequence having at least 90% or at least 95% sequence
identity with residues 53-728 of SEQ ID NO:11 (e.g., at least 96%,
at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%
sequence identity with residues 53-728 of SEQ ID NO:11). In other
variations, the mRNA molecule encodes a PCCB protein comprising an
amino acid sequence having at least 90% or at least 95% sequence
identity with residues 29-539 of SEQ ID NO:13 (e.g., at least 96%,
at least 97%, at least 98%, at least 99%, at least 99.5%, or 100%
sequence identity with residues 29-539 of SEQ ID NO:13). To direct
an encoded MUT, PCCA, PCCB, or BCKDH subunit protein to the
mitochondria of the cell, an mRNA molecule encoding the protein
includes a sequence encoding a mitochondrial leader sequence. The
mitochondrial leader sequence may be that of a native protein
(e.g., residues 1-32 of SEQ ID NO:9 (a native human MUT
mitochondrial leader sequence), residues 1-52 of SEQ ID NO:11 (a
native human PCCA mitochondrial leader sequence), or residues 1-28
of SEQ ID NO:13 (a native human PCCB mitochondrial leader
sequence)), or may be derived from another protein comprising a
mitochondrial targeting signal peptide, or synthesized de novo. An
engineered cleavage site may be included at the junction between
the mitochondrial leader sequence and the remainder of the
polypeptide to optimize proteolytic processing in the cell. The
mitochondrial leader sequence is operably linked to the mRNA
sequence encoding the mature MUT, PCCA, PCCB, or BCKDH subunit
protein, i.e., the two sequences are joined in the correct reading
frame and positioned to direct the newly synthesized polypeptide to
the mitochondria of a cell. In specific variations, the encoded MUT
protein with a mitochondrial leader sequence has an amino acid
sequence as set forth in SEQ ID NO:9, the encoded PCCA protein with
a mitochondrial leader sequence has an amino acid sequence as set
forth in SEQ ID NO: 11, or the encoded PCCB protein with a
mitochondrial leader sequence has an amino acid sequence as set
forth in SEQ ID NO:13. A suitable mRNA sequence encoding a MUT
protein of SEQ ID NO:9, and which may be formulated into a
composition comprising a lipid nanoparticle in accordance with the
present disclosure, may comprise the sequence shown in SEQ ID NO:10
(coding sequence corresponding to residues 48-2297). A suitable
mRNA sequence encoding a PCCA protein of SEQ ID NO:11, and which
may be formulated into a composition comprising a lipid
nanoparticle in accordance with the present disclosure, may
comprise the sequence shown in SEQ ID NO: 12 (coding sequence
corresponding to residues 48-2231). A suitable mRNA sequence
encoding a PCCB protein of SEQ ID NO:13, and which may be
formulated into a composition comprising a lipid nanoparticle in
accordance with the present disclosure, may comprise the sequence
shown in SEQ ID NO:14 (coding sequence corresponding to residues
48-1664). A MUT-, PCCA-, PCCB-, or BCKDH-subunit-encoding mRNA for
formulation with a lipid nanoparticle typically includes a poly(A)
at its 3' end (e.g., a polyA tail of from about 50 to about 500
adenine residues).
[0418] In yet other embodiments comprising increasing the amount of
a protein in a cell the protein is argininosuccinate lyase (ASL) or
argininosuccinate synthetase (ASS1). In such embodiments, an mRNA
encoding an ASL or ASS1 protein is formulated into a lipid
nanoparticle composition and is administered to a subject with
co-injection or separate injection of a membrane-destabilizing
polymer as described herein. In particular variations, the mRNA
molecule encodes an ASL protein comprising an amino acid sequence
having at least 90% or at least 95% sequence identity with SEQ ID
NO:48 (e.g., at least 96%, at least 97%, at least 98%, at least
99%, at least 99.5%, or 100% sequence identity with SEQ ID NO:48).
In other variations, the mRNA molecule encodes an ASS1 protein
comprising an amino acid sequence having at least 90% or at least
95% sequence identity with SEQ ID NO:50 (e.g., at least 96%, at
least 97%, at least 98%, at least 99%, at least 99.5%, or 100%
sequence identity with SEQ ID NO:50). A suitable mRNA sequence
encoding an ASL protein of SEQ ID NO:48, and which may be
formulated into a composition comprising a lipid nanoparticle in
accordance with the present disclosure, may comprise the sequence
shown in SEQ ID NO:49 (coding sequence corresponding to residues
48-1439). A suitable mRNA sequence encoding an ASS1 protein of SEQ
ID NO:50, and which may be formulated into a composition comprising
a lipid nanoparticle in accordance with the present disclosure, may
comprise the sequence shown in SEQ ID NO:51 (coding sequence
corresponding to residues 48-1283). An ASL- or ASS1-encoding mRNA
for formulation with a lipid nanoparticle typically includes a
poly(A) at its 3' end (e.g., a polyA tail of from about 50 to about
500 adenine residues).
[0419] Thus, in certain embodiments of the present invention, an
mRNA is formulated into a lipid nanoparticle as the mRNA carrier.
In some variations, a sequential injection of a
membrane-destabilizing polymer nanoparticle is given approximately
1 to 15 minutes following the mRNA/LNP that enhances delivery of
the mRNA to the cytoplasm in the target cell. In some embodiments
of the present disclosure, the LNP comprises a cationic lipid, a
PEG-lipid, cholesterol, and an anionic lipid. The lipids are
typically solubilized, e.g., in 100% ethanol, typically from 20
mg/mL to 200 mg/mL individually and then mixed together to obtain,
for example, the following lipid ratio ranges: 20-60 mol % cationic
lipid, 0-50 mol % anionic lipid, 0-40 mol % cholesterol, and 0-15
mol % PEG-lipid. A lipid mixture in ethanol is typically prepared
in a range from 1 mg/mL to 40 mg/mL. The mRNA may be prepared using
a standard in vitro transcription reaction according to well-known
procedures. The mRNA solution is typically diluted in an
aqueous/isotonic buffer at about normal physiological pH (e.g., pH
7.4) at a concentration from 0.01 mg/mL to 1 mg/mL. The lipid
mixture in ethanol and mRNA aqueous solution may then be mixed
together at a 1:3 ratio of lipid:mRNA using a microfluidic device.
Lipid concentrations, mRNA concentrations, and mixing ratio can be
adjusted to prepare lipid:mRNA formulations at N:P ratios (nitrogen
to phosphorous ratio between the cationic lipid and the mRNA) from
0.5 to 40. After an incubation time, the mRNA/LNP is typically
dialyzed overnight in an aqueous/isotonic buffer. The polymer may
be solubilized in an aqueous/isotonic buffer at about normal
physiological pH (e.g., pH 7.4). Particularly suitable
concentrations of solubilized polymer range from 1 mg/mL to 50
mg/mL. The formulations may be used for delivery of the mRNA into
target cells (e.g., the formulations may be contacted with cells in
vitro or administered to a subject, such as mice, in vivo).
[0420] In further variations where an mRNA is formulated into a
lipid nanoparticle and delivered in accordance with the present
disclosure, the mRNA/LNP is formulated so as to reduce or eliminate
an undesired immune response in a subject. For example, RNA
transcribed in vitro typically contains multiple contaminants,
including short RNAs produced by abortive initiation events, and
double-stranded (ds)RNAs generated by self-complementary 3'
extension, RNA-primed transcription from RNA templates and
RNA-dependent RNA polymerase activity. See Kariko et al., Nucleic
Acids Research, 2011, 1-10, doi:10.1093/nar/gkr695. These dsRNA
contaminants can be immunostimulatory through binding and
activating a number of innate immune receptors, including toll-like
receptors TLR3, TLR7, TLR8, retinoic acid-inducible gene I (RIG-I),
and RNA-dependent protein kinase (PKR). Further, the presence of
immunostimulatory nucleic acid encapsulated in lipid nanoparticles
containing surface-associated PEG can stimulate an immune response
against the carrier. See Semple et al., J. Pharmacol. Exp. Ther.
312:1020-1026, 2005. Semple et al. showed this immune response to
depend on the presence of non-exchangeable PEG-lipids (DSPE-PEG2000
or PEG ceramide C.sub.20) in the LNP and to lead to rapid plasma
elimination of subsequent repeat administrations of
liposome-encapsulated oligodeoxynucleotide (ODN); in contrast,
nucleic acid encapsulated in a LNP containing an exchangeable
PEG-lipid with a shorter acyl chain (PEG ceramide C.sub.14) showed
no change in circulation levels following repeat administrations.
See Semple et al., supra.
[0421] To reduce or eliminate a potential immune response against
mRNA encapsulated in an LNP, as well as to reduce or eliminate a
potential rapid plasma clearance following repeat administrations
of the mRNA/LNP, certain variations of the mRNA or mRNA/LNP
formulation may be used. For example, the mRNA may be purified
(e.g., using HPLC purification) to remove immunostimulatory dsRNA
contaminants. HPLC-purified mRNA has been shown to avoid
stimulating type I interferon cytokines (IFN-.alpha., IFN-.beta.
and TNF-.alpha.). See Kariko et al., supra. In some variations, one
or more uridines in the mRNA sequence are substituted with
pseudouridine or N1-methyl-pseudouridine, which has been shown to
avoid activating innate immune receptors (see id.). In other
embodiments, the mRNA sequence may be codon optimized to remove or
reduce the number of uridines, which can activate the innate immune
response. In yet other embodiments, an exchangeable PEG-lipid
(e.g., DMPE-PEG2000) in the LNP is used to maintain activity
following repeat administration. Any one or more of these
variations may be used for in vivo delivery of mRNA and related
methods of treatment in accordance with the present disclosure.
[0422] Methods for purifying mRNA are generally known in the art
and may be used to prepare mRNA for formulation with a lipid
nanoparticle in accordance with the present disclosure. For
example, after isolation of in vitro-transcribed (IVT) mRNA
constructs from transcription mixtures, further purification of the
material may be performed using ion-pair/reversed-phase HPLC or
anion-exchange HPLC. These techniques may remove length-based
sequence variants and other nucleic acid impurities when performed
under denaturing conditions. Ion-pair/reversed phase HPLC utilizes
a traditional C8 or C18 stationary phase (alternatively,
polymeric-based media may be used) and a mobile phase system
containing a suitable ion-pairing agent such as triethylammonium
acetate. The material is traditionally eluted using an acetonitrile
gradient. The purification occurs under denaturing conditions
(typically at temperatures >55.degree. C.). Strong or weak
anion-exchange HPLC may also be utilized. For example, a strong
anion exchange column (utilizing a quaternary ammonium in the
stationary phase) may be used with a mobile phase system buffered
at neutral to basic pH (e.g., 20 mM sodium phosphate at pH 8.0),
with elution modulated by gradient addition of a stronger salt
solution (e.g., 1M sodium bromide) to displace interaction of the
nucleic acid backbone with the column stationary phase. Because the
strong ionic environment increases the stability of the mRNA
conformation (and therefore confers a higher Tm relative to the
Ion-pair/reversed phase separations), the purification may require
a higher temperature and/or pH environment to fully melt out
secondary or double-stranded structures.
[0423] In certain embodiments of the present invention, a
therapeutic agent is delivered intracellularly to cells of a target
tissue for treatment of a disease amenable to treatment with the
therapeutic agent. In such embodiments, the therapeutic agent is
delivered to the target tissue via combined administration of a
membrane-destabilizing polymer and lipid nanoparticle comprising
the therapeutic agent as described herein, typically in a manner
otherwise consistent with conventional methodologies associated
with management of the disease or disorder for which treatment is
sought. In accordance with the disclosure herein, a therapeutically
effective amount of the agent is administered to a subject in need
of such treatment for a time and under conditions sufficient to
prevent or treat the disease.
[0424] Subjects for administration of a therapeutic agent as
described herein include patients at high risk for developing a
particular disease as well as patients presenting with an existing
disease. In certain embodiments, the subject has been diagnosed as
having the disease for which treatment is sought. Further, subjects
can be monitored during the course of treatment for any change in
the disease (e.g., for an increase or decrease in clinical symptoms
of the disease).
[0425] In prophylactic applications, pharmaceutical compositions
are administered to a patient susceptible to, or otherwise at risk
of, a particular disease in an amount sufficient to eliminate or
reduce the risk or delay the onset of the disease. In therapeutic
applications, compositions are administered to a patient suspected
of, or already suffering from, such a disease in an amount
sufficient to cure, or at least partially arrest, the symptoms of
the disease and its complications. An amount adequate to accomplish
this is referred to as a therapeutically- or
pharmaceutically-effective dose or amount. In both prophylactic and
therapeutic regimes, agents are usually administered in several
dosages until a sufficient response has been achieved. Typically,
the response is monitored and repeated dosages are given if the
desired response starts to fade.
[0426] To identify subject patients for treatment according to the
methods of the invention, accepted screening methods may be
employed to determine risk factors associated with specific
diseases or to determine the status of an existing disease
identified in a subject. Such methods can include, for example,
determining whether an individual has relatives who have been
diagnosed with a particular disease. Screening methods can also
include, for example, blood tests to assay for buildups of
metabolites caused by missing or mutated proteins in the liver (for
certain liver diseases) or conventional work-ups to determine
familial status for a particular disease known to have a heritable
component (for example, various cancers and protein deficiency
diseases are known to have certain inheritable components).
Inheritable components of cancers include, for example, mutations
in multiple genes that are transforming (e.g., Ras, Raf, EGFR, cMet
and others), the presence or absence of certain HLA and killer
inhibitory receptor (KIR) molecules, or mechanisms by which cancer
cells are able to modulate immune suppression of cells like NK
cells and T cells, either directly or indirectly (see, e.g.,
Ljunggren and Malmberg, Nature Rev. Immunol. 7:329-339, 2007;
Boyton and Altmann, Clin. Exp. Immunol. 149:1-8, 2007). Toward this
end, nucleotide probes can be routinely employed to identify
individuals carrying genetic markers associated with a particular
disease of interest. In addition, a wide variety of immunological
methods are known in the art that are useful to identify markers
for specific diseases. For example, various ELISA immunoassay
methods are available and well-known in the art that employ
monoclonal antibody probes to detect antigens associated with
specific tumors. Screening may be implemented as indicated by known
patient symptomology, age factors, related risk factors, etc. These
methods allow the clinician to routinely select patients in need of
the methods described herein for treatment.
[0427] For administration, a lipid nanoparticle and
membrane-destabilizing polymer are formulated as a single
pharmaceutical composition (for co-injection embodiments; typically
mixed together just prior to administration) or as separate
pharmaceutical compositions (for separate administration
embodiments). A pharmaceutical composition comprising an LNP and/or
membrane-destabilizing polymer can be formulated according to known
methods to prepare pharmaceutically useful compositions, whereby
the LNP and/or polymer component(s) are combined in a mixture with
a pharmaceutically acceptable carrier. A composition is said to be
a "pharmaceutically acceptable carrier" if its administration can
be tolerated by a recipient patient. Sterile phosphate-buffered
saline is one example of a pharmaceutically acceptable carrier.
Other suitable carriers are well-known to those in the art. (See,
e.g., Gennaro (ed.), Remington's Pharmaceutical Sciences (Mack
Publishing Company, 19th ed. 1995).) Formulations may further
include one or more excipients, preservatives, solubilizers,
buffering agents, etc.
[0428] For disease treatment, a pharmaceutical composition is
administered to a subject in a therapeutically effective amount.
According to the methods of the present invention, the lipid
nanoparticle and membrane-destabilizing polymer may be administered
to subjects by a variety of administration modes, including, for
example, by intramuscular, subcutaneous, intravenous, intra-atrial,
intra-articular, parenteral, intranasal, intrapulmonary,
transdermal, intrapleural, intrathecal, and oral routes of
administration. For prevention and treatment purposes, the
compositions may be administered to a subject in a single bolus
delivery, via continuous delivery (e.g., continuous transdermal
delivery) over an extended time period, or in a repeated
administration protocol (e.g., on an hourly, daily, weekly, or
bi-weekly basis).
[0429] Determination of the proper dosage for a particular
situation is within the skill in the art. Determination of
effective dosages in this context is typically based on animal
model studies followed up by human clinical trials and is guided by
determining effective dosages and administration protocols that
significantly reduce the occurrence or severity of the subject
disease in model subjects. Effective doses of the compositions of
the present invention vary depending upon many different factors,
including means of administration, target site, physiological state
of the patient, whether the patient is human or an animal, other
medications administered, whether treatment is prophylactic or
therapeutic, as well as the specific activity of the composition
itself and its ability to elicit the desired response in the
individual. Usually, the patient is a human, but in some diseases,
the patient can be a nonhuman mammal. Typically, dosage regimens
are adjusted to provide an optimum therapeutic response, i.e., to
optimize safety and efficacy. Accordingly, a therapeutically or
prophylactically effective amount is also one in which any
undesired collateral effects are outweighed by beneficial effects.
For administration of a therapeutic agent, a dosage typically
ranges from about 0.1 .mu.g to about 100 mg/kg or about 1 .mu.g/kg
to about 50 mg/kg, and more usually about 1 .mu.g/kg to about 10
mg/kg or about 10 .mu.g to about 5 mg/kg of the subject's body
weight, exclusive of other LNP components. In more specific
embodiments, an effective amount of the agent is between about 1
.mu.g/kg and about 20 mg/kg, between about 10 .mu.g/kg and about 10
mg/kg, or between about 0.1 mg/kg and about 5 mg/kg, exclusive of
other LNP component. The quantity of a membrane-destabilizing
polymer may be varied or adjusted, for example, from about 10 .mu.g
to about 200 mg/kg, about 10 .mu.g to about 100 mg/kg, about 0.1
mg/kg to about 100 mg/kg, about 0.1 mg/kg to about 50 mg/kg, or
about 0.5 mg/kg to about 50 mg/kg. Dosages within this range can be
achieved by single or multiple administrations, including, e.g.,
multiple administrations per day or daily, weekly, bi-weekly, or
monthly administrations. For example, in certain variations, a
regimen consists of an initial administration followed by multiple,
subsequent administrations at weekly or bi-weekly intervals.
Another regimen consists of an initial administration followed by
multiple, subsequent administrations at monthly or bi-monthly
intervals. Alternatively, administrations can be on an irregular
basis as indicated by monitoring of physiological correlates of the
disease and/or clinical symptoms of the disease.
[0430] Lipid nanoparticles can adsorb to virtually any type of cell
and then slowly release the encapsulated agent. Alternatively, an
absorbed lipid nanoparticle may be endocytosed by cells (e.g.,
cells that are phagocytic). Endocytosis is typically followed by
intralysosomal degradation of LNP lipids and release of the
encapsulated agents (see Scherphof et al., Ann. N.Y. Acad. Sci.
446:368, 1985). After intravenous administration, lipid
nanoparticles (e.g., liposomes of about 0.1 to 1.0 .mu.m) are
typically taken up by cells of the reticuloendothelial system,
located principally in the liver and spleen. This preferential
uptake of smaller liposomes by the cells of the reticuloendothelial
system has been used to deliver chemotherapeutic agents to
macrophages and to tumors of the liver. As described herein, it is
believed the combining administration of a lipid nanoparticle
together with administration of a membrane-destabilizing polymer
enhances efficiency of delivery of the LNP-associated therapeutic
agent to the cytosol of a cell.
[0431] The reticuloendothelial system can be circumvented by
several methods including saturation with large doses of lipid
nanoparticles, or selective macrophage inactivation by
pharmacological means (see Claassen et al., Biochim. Biophys. Acta
802:428, 1984). In addition, incorporation of glycolipid- or
polyethelene glycol-derivatized phospholipids into liposome
membranes has been shown to result in a significantly reduced
uptake by the reticuloendothelial system (see Allen et al.,
Biochim. Biophys. Acta 1068:133, 1991; Allen et al., Biochim.
Biophys. Acta 1150:9, 1993).
[0432] Lipid nanoparticles can also be prepared to target
particular cells or tissues by varying phospholipid composition of
the lipid nanoparticles. For example, liposomes prepared with a
high content of a nonionic surfactant have been used to target the
liver. (See, e.g., Japanese Patent 04-244,018 to Hayakawa et al.;
Kato et al., Biol. Pharm. Bull. 16:960, 1993.) These formulations
were prepared by mixing soybean phospatidylcholine,
.alpha.-tocopherol, and ethoxylated hydrogenated castor oil
(HCO-60) in methanol, concentrating the mixture under vacuum, and
then reconstituting the mixture with water. A liposomal formulation
of dipalmitoylphosphatidylcholine (DPPC) with a soybean-derived
sterylglucoside mixture (SG) and cholesterol (Ch) has also been
shown to target the liver. (See Shimizu et al., Biol. Pharm. Bull.
20:881, 1997.)
[0433] Lipid nanoparticles and/or membrane-destabilizing polymers
can also be prepared to target particular cells or tissues by using
a targeting ligand as discussed herein.
[0434] In some embodiments, a lipid nanoparticle and
membrane-destabilizing polymer as described herein are used in a
method for treating a disease associated with defective gene
expression and/or activity in a subject. Such methods of treatment
include administering to a subject having the disease associated
with defective gene expression and/or activity (a) an effective
amount of a lipid nanoparticle comprising a polynucleotide that is
homologous to and can silence, for example by cleavage, a gene or
that specifies the amino acid sequence of a protein and is
translated during protein synthesis, and (b) an effective amount of
a membrane-destabilizing polymer, where the polynucleotide is
delivered into the cytosol of target cells of a target tissue
associated with the disease, thereby treating the disease. In some
embodiments, at least one of the lipid nanoparticle and
membrane-destabilizing polymer includes a targeting ligand that
specifically binds to a molecule on the surface of the target cells
of the target tissue within the subject. Examples of a disease
associated with defective gene expression and/or activity in a
subject treatable by the methods disclosed herein include liver
cancer (e.g., hepatocellular carcinoma), hepatitis,
hypercholesterolemia, liver fibrosis, and haemochromatosis. In
other variations, a disease or condition associated with defective
gene expression and/or activity in a subject treatable by the
methods disclosed herein is a cancer of the breast, ovaries,
pancreas, endometrium, lungs, kidneys, colon, brain (e.g.,
glioblastoma), or myeloid cells of hematopoietic origin.
[0435] In certain embodiments, the disease associated with
defective gene expression is a disease characterized by a
deficiency in a functional polypeptide (also referred to herein as
a "disease associated with a protein deficiency" or a "protein
deficiency disease"). Such methods of treatment include
administering to a subject having the protein deficiency disease
(a) an effective amount of a lipid nanoparticle comprising an mRNA
that encodes the functional protein or a protein having the same
biological activity as the functional protein and (b) an effective
amount of a membrane-destabilizing polymer, where the mRNA is
delivered into the cytosol of target cells of a target tissue
associated with the protein deficiency, and where the mRNA is
translated during protein synthesis so as to produce the encoded
protein within the target tissue in an amount sufficient to treat
the disease. In some embodiments, at least one of the lipid
nanoparticle and membrane-destabilizing polymer comprises a
targeting ligand that specifically binds to a molecule on the
surface of the target cells of the target tissue. In specific
variations, the mRNA encodes a functional erythropoietin,
alpha-galactosidase A, LDL receptor, Factor VII, Factor VIII,
Factor IX, alpha-L-iduronidase, iduronate-2-sulfatase,
heparan-N-sulfatase, alpha-N-acetylglucosaminidase, galactose
6-sulfatase, acid (3-galactosidase, lysosomal acid lipase,
ornithine transcarbamylase (OTC), alpha-1-antitrypsin,
arylsulfatase A, arylsulfatase B, acid ceramidase, acid
.alpha.-L-fucosidsase, acid .beta.-glucosidase (also known as
glucocerebrosidase), galactocerebrosidase, acid
.alpha.-mannosidase, acid .beta.-mannosidase,
N-acetylgalactosamine-6-sulfate sulfatase, acid sphingomyelinase,
acid .alpha.-glucosidase, 3-hexosaminidase B,
acetyl-CoA:.alpha.-glucosaminide N-acetyltransferase,
N-acetylglucosamine-6-sulfate sulfatase,
alpha-N-acetylgalactosaminidase, sialidase, .beta.-glucuronidase,
or .beta.-hexosaminidase A. In other embodiments, the mRNA encodes
a functional Retinoblastoma protein (pRb), p53 tumor-suppressor
protein, Phosphatase and tensin homolog (PTEN), Von Hippel-Lindau
tumor suppressor (pVHL), Adenomatous polyposis coli (APC), FAS
receptor (FasR), Suppression of tumorigenicity 5 (ST5), YPEL3,
Suppressor of tumorigenicity protein 7 (ST7), or Suppressor of
tumorigenicity 14 protein (ST14). In yet other embodiments, the
mRNA encodes a functional Galactose-1-phosphate
uridylyltransferase, Galactokinase, UDP-galactose 4-epimerase,
Transthyretin, complement regulatory protein (e.g., factor H,
factor I, or membrane cofactor protein), phenylalanine hydroxylase
(PAH), homogentisate 1,2-dioxygenase, Porphobilinogen deaminase,
hypoxanthine-guanine phosphoribosyltransferase (HGPRT),
argininosuccinate lyase (ASL), argininosuccinate synthetase (ASS1),
or P-type ATPase protein, FIC-1.
[0436] Further examples of a disease or condition associated with
defective gene expression and/or activity in a subject treatable by
the methods disclosed herein include protein deficiency diseases
associated with single-gene metabolic defects in the liver.
Exemplary protein deficiency diseases of the liver include diseases
associated with urea cycle defects (e.g., ornithine
transcarbamylase (OTC) deficiency, carbamoyl phosphate synthetase I
(CPS1) deficiency, argininosuccinic aciduria (argininosuccinate
lyase (ASL) deficiency), and citrullinemia (argininosuccinate
synthetase (ASS1) deficiency)); tyrosinemia type 1
(fumarylacetoacetase (FAH) enzyme deficiency); primary
hyper-oxaluria type 1 (alanine:glyoxylate-aminotransferase (AGT)
deficiency); organic acidemia (e.g., methylmalonic acidemia (MMA;
deficiency in, for example, methylmalonyl CoA mutase), propionic
acidemia (PA; propionyl CoA carboxylase (PCC) deficiency), and
maple syrup urine disease (MSUD; branched-chain ketoacid
dehydrogenase (BCKDH) deficiency)); Wilson's Disease (deficiency in
copper-transporting ATPase, Atp7B); Crigler-Najjar Syndrome Type 1
(bilirubin uridinediphosphate glucuronyltransferase (BGT) enzyme
deficiency); hemochromatosis (hepcidin deficiency); glycogen
storage disease (GSD) type 1a (glucose-6-phosphatase (G6Pase)
deficiency); glycogen storage disease (GSD) type 1b (glucose
6-phosphate translocase deficiency); lysosomal storage diseases
(LSDs; deficiencies in lysosomal enzymes) such as, e.g., Gaucher's
Disease types 1, 2, and 3 (lysosomal glucocerebrosidase (GB)
deficiency), Niemann-Pick Disease Type C (mutation in either the
NPC1 or NPC2 gene), and Niemann-Pick Disease Types A and B (acid
sphingomyelinase (ASM) deficiency); alpha-1 antitrypsin (A1AT)
deficiency; hemophilia B (Factor IX deficiency); galactosemia types
1, 2, and 3 (galactose-1-phosphate uridylyltransferase,
galactokinase, and UDP-galactose 4-epimerase deficiencies,
respectively); transthyretin-related hereditary amyloidosis
(TTR-familial amyloid polyneuropathy; transthyretin deficiency);
atypical haemolytic uremic syndrome-1 (deficiencies in complement
regulatory proteins, e.g., factor H, factor I, or membrane cofactor
protein); phenylketonuria (phenylalanine hydroxylase (PAH)
deficiency); alcaptonuria (homogentisate 1,2-dioxygenase
deficiency); acute intermittent porphyria (porphobilinogen
deaminase deficiency); Lesch-Nyhan syndrome (hypoxanthine-guanine
phosphoribosyltransferase (HGPRT) deficiency; and progressive
familial intrahepatic cholestasis (PFIC) (P-type ATPase protein,
FIC-1 deficiency). Additional examples of protein deficiency
diseases that are lysosomal storage diseases (LSDs) include Fabry
disease (alpha-galactosidase A deficiency); Farber disease (acid
ceramidase deficiency); fucosidosis (acid .alpha.-L-fucosidsase
deficiency); GM1 gangliosidosis (acid .beta.-galactosidase
deficiency); Hunter syndrome (mucopolysaccharidosis type II (MPS
II); iduronate-2-sulfatase deficiency); Hurler-Scheie, Hurler, and
Scheie syndromes (mucopolysaccharidosis type I (MPS I);
alpha-L-iduronidase deficiency); Krabbe disease
(galactocerebrosidase deficiency); .alpha.-mannosidosis (acid
.alpha.-mannosidase deficiency); .beta.-mannosidosis (acid
.beta.-mannosidase deficiency); Maroteaux-Lamy syndrome
(mucopolysaccharidosis type VI (MPS VI); arylsulfatase B
deficiency); metachromatic leukodystrophy (arylsulfatase A
deficiency); Morquio syndrome type A (mucopolysaccharidosis type
IVA (MPS IVA); N-acetylgalactosamine-6-sulfate sulfatase
deficiency); Morquio syndrome type B (mucopolysaccharidosis type
IVB (MPS IVB); acid f-galactosidase deficiency); Pompe disease
(acid .alpha.-glucosidase deficiency); Sandhoff disease
(.beta.-hexosaminidase B deficiency); Sanfilippo syndrome type A
(mucopolysaccharidosis type IIIA (MPS IIIA); heparan-N-sulfatase
deficiency); Sanfilippo syndrome type B (mucopolysaccharidosis type
IIIB (MPS IIIB); alpha-N-acetylglucosaminidase deficiency);
Sanfilippo syndrome type C (mucopolysaccharidosis type IIIC (MPS
IIIC); acetyl-CoA:.alpha.-glucosaminide N-acetyltransferase
deficiency); Sanfilippo syndrome type D (mucopolysaccharidosis type
IIID (MPS IIID); N-acetylglucosamine-6-sulfate sulfatase
deficiency); Schindler/Kanzaki disease
(alpha-N-acetylgalactosaminidase deficiency); sialidosis (sialidase
deficiency); Sly syndrome (mucopolysaccharidosis type VII (MPS
VII); .beta.-glucuronidase deficiency); and Tay-Sachs disease
(.beta.-hexosaminidase A deficiency).
[0437] In particular variations, an mRNA encoding an ornithine
transcarbamylase (OTC) protein is delivered in accordance with the
present methods to treat ornithine transcarbamylase deficiency
(OTCD). OTCD is a urea cycle disorder that can trigger
hyperammonemia, a life-threatening illness that leads to brain
damage, coma or even death. This is due to deficiency in the
activity of OTC, a key enzyme in the urea cycle, which primarily
takes place in the liver and is responsible for removal of excess
nitrogen in the body. Ammonium nitrogen is produced from protein
intake as well as protein breakdown in the body. In the liver, this
ammonium nitrogen is converted into urea by enzymes in the urea
cycle. Urea is non-toxic and cleared easily through the kidneys in
urine, normally. However, when the OTC enzyme is deficient, ammonia
levels rise in blood and cause severe brain damage. Patients with
severe OTC deficiency are most often identified 2-3 days after
birth where the patient has significantly elevated blood ammonia
levels and ends up in a coma. Patients with milder OTC deficiency
can have crises during times of stress resulting in elevated
ammonia levels that can also lead to coma. Current therapies
include ammonia scavenger drugs (Buphenyl, Ravicti) for use in
patients with hyperammonemia.
[0438] The OTC gene is X-linked. The disease is present in males
with one mutant allele and in females either homozygous or
heterozygous with mutant alleles. Male patients are typically those
with the severest OTC deficiency found right after birth. In
addition to elevation in blood ammonia levels, urinary orotic acid
levels are also elevated. In patients with severe OTC deficiency,
OTC enzyme activity is <20% of normal levels. In patients with
milder OTC deficiency, OTC enzyme activity is up to 30% of normal
levels.
[0439] A method for treating OTCD with a lipid nanoparticle
comprising an OTC-encoding mRNA and a membrane-destabilizing
polymer generally includes administering to a subject having OTCD
an effective amount of the lipid nanoparticle and an effective
amount of the membrane-destabilizing polymer, where at least one of
the lipid nanoparticle and membrane-destabilizing polymer includes
a targeting ligand that specifically binds to a molecule on the
surface of liver cells within the subject, and whereby the
OTC-encoding mRNA is delivered to liver cells and translated during
protein synthesis to produce the OTC protein. The OTC-encoding mRNA
may be an mRNA as set forth above with respect to a method for
increasing OTC protein in a cell.
[0440] The efficacy of a composition or method for treating a
disease can be evaluated in vivo in animal models of disease.
Particularly suitable animal models for evaluating efficacy of a
[lipid nanoparticle]/[membrane-destabilizing polymer] composition
(or combination of LNP composition and polymer composition) for
treatment of OTCD includes known mouse models having deficiencies
of the OTC enzyme in the liver. One such mouse model,
OTC-spf.sup.ash (sparse fur and abnormal skin and hair) mice,
contain an R129H mutation resulting in reduced levels of OTC
protein and have only 5-10% of the normal level of enzyme activity
in liver (see Hodges et al., Proc. Natl. Acad. Sci. USA
86:4142-4146, 1989). Another model, OTC-spf mice, contain an H117N
mutation which results in reduced levels of enzyme activity to
5-10% of normal levels (see Rosenberg et al., Science 222:426-428,
1983). Both of these mouse models have elevated urine orotic acid
levels compared to their wild-type littermate mice. A third model
for OTC deficiency is inducing hyperammonemia in OTC-spf or
OTC-spf.sup.ash mice (Cunningham et al., Mol Ther 19: 854-859,
2011). These mice are treated with OTC siRNA or AAV2/8 vector/OTC
shRNA to knockdown residual endogenous OTC expression and activity.
Plasma ammonia levels are elevated and mice die within
approximately 7-28 days.
[0441] In additional variations, an mRNA encoding an enzyme
deficient in an organic acidemia is delivered in accordance with
the present methods to treat the organic acidemia. Organic acidemia
(also known as aciduria) (OA) is a group of disorders characterized
by the excretion of non-amino organic acids in the urine. Most
organic acidemias result from dysfunction of a specific step in
amino acid catabolism, usually the result of deficient enzyme
activity. The majority of organic acid disorders are caused by
abnormal amino acid catabolism of branched-chain amino acids or
lysine. They include propionic acidemia (PA), methylmalonic
acidemia (MMA), maple syrup urine disease (MSUD), and others. These
organic acidemias are inherited in an autosomal recessive manner. A
neonate affected with an OA is usually well at birth and for the
first few days of life. The usual clinical presentation is that of
toxic encephalopathy and includes vomiting, poor feeding,
neurologic symptoms such as seizures and abnormal tone, and
lethargy progressing to coma. Outcome can be improved by diagnosis
and treatment in the first ten days of life. In the older child or
adolescent, variant forms of the OAs can present as loss of
intellectual function, ataxia or other focal neurologic signs, Reye
syndrome, recurrent ketoacidosis, or psychiatric symptoms.
[0442] Clinical laboratory findings indicate that organic acidemias
include acidosis, ketosis, hyperammonemia, abnormal liver function,
hypoglycemia, and neutropenia. First-line diagnosis in the organic
acidemias is urine organic acid analysis using gas chromatography
with mass spectrometry (GC/MS). The urinary organic acid profile is
nearly always abnormal in the face of acute illness. Confirmatory
testing involves assay of the activity of the deficient enzyme in
lymphocytes or cultured fibroblasts and/or molecular genetic
testing. Characteristics of the three primary disorders are
summarized in Table 1.
TABLE-US-00003 TABLE 1 Metabolic Findings in Organic Acidemias
Caused by Abnormal Amino Acid Catabolism Diagnostic Analytes by
GC/MS and Amino Acid Quantitative Amino Disorder Pathway(s)
Affected Enzyme Acid Analysis Propionic acidemia Isoleucine,
valine, Propionyl CoA Propionic acid, 3-OH (PA) methionine,
threonine carboxylase (PCC) propionic acid, methyl (composed of
three citric acid, propionyl PCCA subunits and glycine in urine
three PCCB subunits) Propionyl carnitine, increased glycine in
blood Methylmalonic Isoleucine, valine, Methylmalonyl CoA
Methylmalonic acid in acidemia (MMA) methionine, threonine mutase
(MUT) blood and urine Propionic acid, 3-OH propionic acid, methyl
citrate in urine Acyl carnitines, increased glycine in blood Maple
syrup urine Leucine, isoleucine, Branched-chain Branched-chain
disease (MSUD) valine ketoacid ketoacids and dehydrogenase
hydroxyacids in urine (BCKDH) Alloisoleucine in (composed of four
plasma different subunits)
[0443] Once the detection of specific analytes narrows the
diagnostic possibilities, the activity of the deficient enzyme is
assayed in lymphocytes or cultured fibroblasts as a confirmatory
test. For many pathways, no single enzyme assay can establish the
diagnosis. For others, tests such as complementation studies need
to be done.
[0444] The goal of therapy is to restore biochemical and
physiologic homeostasis. Neonates require emergency diagnosis and
treatment depending on the specific biochemical lesion, the
position of the metabolic block, and the effects of the toxic
compounds. Treatment strategies include: (1) dietary restriction of
the precursor amino acids and (2) use of adjunctive compounds to
(a) dispose of toxic metabolites or (b) increase activity of
deficient enzymes. Liver transplantation has been successful in a
small number of affected individuals. Even with current clinical
management approaches, individuals with organic acidemias have a
greater risk of infection and a higher incidence of pancreatitis,
which can be fatal.
[0445] Enzyme replacement therapy via specific mRNA delivery to the
liver offers the most effective treatment of the organic acidemias.
In certain embodiments of a method for treating an organic
acidemia, an mRNA encoding a methylmalonyl CoA mutase (MUT) is
delivered to a subject in accordance with the present methods to
treat methylmalonic acidemia MMA. In other embodiments, an mRNA
encoding a PCC subunit (PCCA or PCCB) is delivered to a subject in
accordance with the present methods to treat propionic acidemia
(PA). In yet other embodiments, an mRNA encoding a BCKDH subunit is
delivered to a subject in accordance with the present methods to
treat maple syrup urine disease (MSUD). A method for treating MMA,
PA, or MSUD with a lipid nanoparticle comprising an Mut, Pcca/b, or
BCKDH subunit mRNA and a membrane-destabilizing polymer generally
includes administering to a subject having an organic acidemia of
the specified type an effective amount of the lipid nanoparticle
and an effective amount of the membrane-destabilizing polymer,
where at least one of the lipid nanoparticle and
membrane-destabilizing polymer includes a targeting ligand that
specifically binds to a molecule on the surface of liver cells
within the subject, and whereby the Mut, Pcca/b, or BCKDH subunit
mRNA is delivered to liver cells and translated during protein
synthesis to produce the respective protein. A Mut or Pcca/b mRNA
may be an mRNA as set forth above with respect to a method for
increasing the respective protein in a cell.
[0446] The efficacy of a composition or method for treating an
organic acidemia disease can be evaluated in vivo in animal models
of disease. For example, particularly suitable animal models for
evaluating efficacy of a mRNA/LNP and polymer composition (or
combination of mRNA/LNP composition and polymer composition) for
treatment of MMA and PA are as follows. Mut.sup.-/- neonatal mice
with a severe form of MMA, which normally die within the first 21
days of life, have been successfully treated with
hepatocyte-directed delivery of the methylmalonyl-CoA mutase (Mut)
gene. Following an intrahepatic injection of adeno-associated virus
expressing the murine Mut gene, Mut.sup.-/- mice were rescued and
lived beyond 1 year of age (Carrillo-Carrasco et al., Hum. Gene
Ther. 21:1147-1154, 2010). Another MMA disease model where mice
survive into adulthood is Mut.sup.-/- mice with Mut cDNA expressed
under the control of an insulated, muscle-specific promoter
(Mut.sup.-/-;Tg.sup.INS-MCK-Mut) (Manoli et al., 2011, SIMD
Abstract). These mice have elevated plasma methylmalonic acid
levels and decreased oxidative capacity as measured by a .sup.13C
propionate oxidation/breathe assay. A mouse model of PA
(Pcca.sup.-/- mice) succumbs to death 24-36 h after birth and is
associated with fatal ketoacidosis (Miyazaki et al., J. Biol. Chem.
276:35995-35999, 2001). Pcca gene transfer that provides a
postnatal PCC activity of 10-20% in the liver of a transgenic mouse
strain attenuates the fatal ketoacidosis in newborn mice (Miyazaki
et al., 2001, supra). Recently, an intrahepatic adeno-associated
virus mediated gene transfer for human Pcca was tested in neonatal
Pcca.sup.-/- mice (Chandler et al., Hum. Gene Ther. 22:477-481,
2010). The authors found a sustained therapeutic effect as
demonstrated in a survival rate of approximately 64% and reduction
of disease-related metabolites (Chandler et al., 2010, supra).
Another mouse disease model of PA is a hypomorphic model where
Pcca.sup.-/- mice express a transgene bearing an A138T mutant of
the PCCA protein. These mice have 2% of wild-type PCC activity,
survive to adulthood and have elevations in disease-related
metabolites (Guenzel et al., Mol. Ther. 21:1316-1323, 2013).
Treatment of these mice with adeno-virus or AAV vector expressing
human PCCA cDNA resulted in increased PCC enzyme activity and
correction of disease marker levels (Guenzel et al., 2013, supra).
Taken together, in murine models of MMA and PA gene transfer
approaches rescue neonatal mice or restore enzyme activity and
correct disease metabolite levels in adult disease models thereby
permitting evaluation of mRNA delivery for restoration of the
defective enzymes.
[0447] In additional variations, an mRNA encoding arginosuccinate
lyase (ASL) or argininosuccinate synthetase (ASS1) is delivered in
accordance with the present methods to treat argininosuccinate
aciduria (ASA) or citrullinemia type I (CTLN I), respectively.
Suitable animal models for evaluating efficacy of a mRNA/LNP and
polymer for treatment of ASA and CTLN I are as follows. ASL
hypomorphic mice have a neomycin gene inserted into intron 9 which
leads to deficiency in the ASL enzyme (.about.10% of wild type
levels of mRNA and protein) and elevations in argininosuccinate and
citrulline plasma levels (Erez et al., Nat Med. 17:1619-1626, 2011)
which is the signature of ASA. These mice if left untreated will
die on their own starting around 3 weeks of age. Treatment of these
mice with helper dependent adenoviral vector expressing mouse ASL
at 4 weeks of age led to improved survival, normalized ASL protein
expression, and reduction in argininosuccinate and citrulline
plasma levels (Nagamani et al., Am J Hum Genet. 90:836-846, 2012).
ASS1 hypomorphic mice result from a spontaneous recessive mutation
(T389I substitution) known as follicular dystrophy (fold). This
mutation leads to unstable ASS1 protein structure and .about.5-10%
of normal enzyme activity. Homozygous fold/fold mice have elevated
plasma citrulline and ammonia levels. These mice will also die on
their own if untreated (Perez et al., Am J Pathol. 177:1958-1968,
2010). Treatment of these mice with AAV8 vector expressing human
ASS1 led to improved survival and decreased plasma citrulline and
ammonia levels (Chandler et al., Gene Ther. 20:1188-1191, 2013).
Thus, in murine models of ASA and CTLN I hepatic gene transfer
methods restore enzyme activity and correct the disease thereby
permitting evaluation of mRNA delivery for restoration of the
defective enzymes.
[0448] In certain other embodiments of a method of treating a
disease associated with defective gene expression and/or activity,
the gene is selected from a growth factor gene, a growth factor
receptor gene, a gene encoding an enzyme (for example, a
phosphatase or a kinase, e.g., a protein tyrosine, serine, or
threonine kinase), an adaptor protein gene, a gene encoding a G
protein superfamily molecule, or a gene encoding a transcription
factor.
[0449] Further examples of suitable gene targets useful in the
methods of treating a disease associated with defective gene
expression and/or activity as described herein include the
following genes or genes encoding the following proteins: MEX3,
MMP2, ApoB, ERBB2, Vascular Endothelial Growth Factor (VEGF),
Vascular Endothelial Growth Factor Receptor (VEGFR), Platelet
Derived Growth Factor Receptor (PDGF), ABL, KITT, FMS-like tyrosine
kinase 3 (FLT3), Cay-1, Epidermal Growth Factor Receptor (EGFR),
H-Ras, K-Ras, N-Ras, Bcl-2, Survivin, FAK, STAT-3, HER-3,
Beta-Catenin, ornithine transcarbamylase, alpha-1-antitrypsin, and
Src.
[0450] Other examples of suitable gene targets useful in the
methods of treating a disease associated with defective gene
expression and/or activity as described herein include tumor
suppressors, where loss of function of the mutated gene can be
corrected by delivery of mRNA encoding the functional protein to
treat cancer. Suitable tumor suppressor targets include
Retinoblastoma protein (pRb), p53 tumor-suppressor protein,
Phosphatase and tensin homolog (PTEN), Von Hippel-Lindau tumor
suppressor (pVHL), Adenomatous polyposis coli (APC), FAS receptor
(FasR), Suppression of tumorigenicity 5 (ST5), YPEL3, Suppressor of
tumorigenicity protein 7 (ST7), and Suppressor of tumorigenicity 14
protein (ST14).
[0451] In certain embodiments, a membrane-destabilizing polymer and
a lipid nanoparticle comprising a therapeutic agent as described
herein is used in the preparation of a medicament or combination of
medicaments for the treatment of a disease amenable to treatment
with the therapeutic agent. In some such embodiments, the disease
is a disease associated with defective gene expression and/or
activity in a subject.
[0452] In some embodiments, a membrane-destabilizing polymer and a
lipid nanoparticle comprising an mRNA encoding a functional protein
as described herein is used in the preparation of a medicament or
combination of medicaments for the treatment of a disease
associated with deficiency in a functional protein.
[0453] The invention is further illustrated by the following
non-limiting examples.
Examples
[0454] Throughout this description, various known acronyms and
abbreviations are used to describe monomers or monomeric residues
derived from polymerization of such monomers. Without limitation,
unless otherwise noted: "BMA" (or the letter "B" as equivalent
shorthand notation) represents butyl methacrylate or monomeric
residue derived therefrom; "DMAEMA" (or the letter "D" as
equivalent shorthand notation) represents N,N-dimethylaminoethyl
methacrylate or monomeric residue derived therefrom; "PAA" (or the
letter "P" as equivalent shorthand notation) represents
2-propylacrylic acid or monomeric residue derived therefrom;
"PEGMA.sub.n", wherein n=8-9 or 4-5, refers to the pegylated
methacrylic monomer,
CH.sub.3O(CH.sub.2CH.sub.2O).sub.nC(O)C(CH.sub.3)CH.sub.2 or
monomeric residue derived therefrom; "PDSMA" represents
2-(pyridin-2-yldisulfanyl)ethyl methacrylate or monomeric residue
derived therefrom; "TFPMA" represents 2,3,5,6-tetrafluorphenyl
methacrylate or monomeric residue derived therefrom; "PFPMA"
represents pentafluorophenyl methacrylate or monomeric residue
derived therefrom. In each case, any such designation indicates the
monomer (including all salts, or ionic analogs thereof), or a
monomeric residue derived from polymerization of the monomer
(including all salts or ionic analogs thereof), and the specific
indicated form is evident by context to a person of skill in the
art. Figures of polymers or macro CTAs in the following examples
are not meant to describe any particular arrangement of the
constitutional units within a particular block. "KDa" and "k" as
used herein refer to molecular weight in kilodaltons.
[0455] The following figure is illustrative of the structures of
the monomers used in the preparation of the polymers:
##STR00011## ##STR00012## ##STR00013## ##STR00014##
[0456] .sup.1H NMR spectra of the monomers and polymers were
recorded on a Varian 400 MHz in deuterated solvents at 25.degree.
C.
[0457] Mass spectra was acquired on Bruker Esquire Ion Trap
instrument using the following settings: electro-spray ionization,
capillary exit voltage of 100.0 V, scanning from 80.00 m/z to
2200.00 m/z, dry gas flow of 6.0 L/min. Mass spectroscopy was also
conducted on a 6520 Accurate Mass Q-TOF LC/MS equipped with an
Agilent 1290 Infinity UHPLC system with UV detector.
[0458] Gel permeation chromatography (GPC) was used to determine
molecular weights and polydispersities (PDI, M.sub.w/M.sub.n) of
the copolymer samples in DMF using a Viscotek GPCmax VE2001 and
refractometer VE3580 (Viscotek, Houston, Tex.). Analysis was
conducted using two PolarGel-M columns (300 mm.times.7.5 mm,
Agilent Technologies) with matching guard column in series at
57.degree. C., or two PolarGel-L columns (300 mm.times.7.5 mm,
Agilent Technologies) with matching guard column in series at
57.degree. C., or two TSKgel G3000SW columns (300 mm.times.7.5 mm,
10 .mu.m, Tosoh Biosciences LLC) in series at 57.degree. C.
HPLC-grade dimethylformamide (DMF) containing 1.0 wt % LiBr was
used as the mobile phase.
[0459] UV/Vis spectroscopy was performed using a NanoDrop UV/Vis
spectrometer (path length 0.1 cm).
[0460] Particle sizes of the polymers were measured by dynamic
light scattering using a Malvern Zetasizer Nano ZS.
[0461] HPLC analysis was performed on Shimadzu LD-20AB with the
variable-wavelength UV detector with a C18 analytical reverse phase
column (ES Industries Chromega Columns, Sonoma C18 catalog number
155B21-SMA-C18(2), 100 .ANG., 25.0 cm.times.4.6 mm, column heated
to 30.degree. C., or a C18 Phenomenex 5.mu. 100 .ANG. 250.times.4.6
mm.times.5 micron (Part#00G-4252-E0) Luna column with guard column
heated to 30.degree. C.).
[0462] All reagents were from commercial sources, unless indicated
otherwise, and the monomers were purified from traces of
stabilizing agents prior to use in the polymerization reactions.
Cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT)
was obtained from Omm Scientific. Azobisisobutyronitrile (AIBN)
(Wako chemicals) was used as the radical initiator in all
polymerization reactions, unless stated otherwise.
Example 1: Lipid mRNA Nanoparticle Formulation with Sequential
Injection of a Polymer
[0463] DOTAP (Corden Pharma, Boulder, Colo., USA; catalog number
LP-R4-117) or DOTMA (Avanti Polar Lipid Alabaster, Ala., USA;
catalog number 890898P) was solubilized at 200 mg/mL in 200 proof
ethanol at room temperature for 15 minutes. The DMPE-PEG.sub.2K
(Corden Pharma, Boulder, Colo., USA; catalog number LP-R4-123) was
solubilized at 25 mg/mL in 200 proof ethanol at room temperature
for 15 minutes. The cholesteryl hemisuccinate (CHEMS) (Avanti Polar
Lipid Alabaster, Ala., USA; catalog number 850524P) and the
Cholesterol (CHOL) (Corden Pharma, Boulder, Colo., USA; catalog
number CH-0355) were individually solubilized at 25 mg/mL in 200
proof at 75.degree. C. for 5 minutes. Typically, for a 2 mL
preparation of a DOTAP:CHEMS:CHOL:DMPE-PEG.sub.2K (50:32:16:2 mol
%) LNP at N:P ratio of 7, a lipid ethanolic mixture containing 22
.mu.L of DOTAP at 200 mg/mL in 200 proof ethanol, 79 .mu.L of CHEMS
at 25 mg/mL in 200 proof ethanol, 31.4 .mu.L of CHOL at 25 mg/mL in
200 proof ethanol, 27.4 .mu.L of DMPE-PEG.sub.2K at 25 mg/mL in 200
proof ethanol and 506 .mu.L of 200 proof ethanol was prepared for a
final volume of 0.666 mL and a final lipid concentration of 11.83
mg/mL. The lipid nanoparticle (LNP) formulations were prepared at
N:P (nitrogen to phosphate) ratios from 3.5 to 28 based on the
DOTAP or DOTMA concentration. The DOTAP:CHEMS or DOTMA:CHEMS ratio
was fixed at 1.6 at 50:32 mol % respectively at the various N:P
ratios. DMPE-PEG.sub.2K was varied from 2 to 5 mol %. The CHOL mol
% was adjusted to result in 100 mol % final lipid
concentration.
[0464] The Fluc (firefly luciferase) mRNA stock solution at 1 mg/mL
in 10 mM Tris-HCl (pH 7.5) (TriLink Biotechnologies, San Diego,
Calif., USA; catalog number L-6107) was diluted to 0.225 mg/mL in
20 mM HEPES/5% glucose, pH 7.4 buffer (HEPES buffer). The mRNA/LNPs
were assembled at N:P ratios from 3.5 to 28 by mixing the ethanolic
lipid solution with 0.225 mg/mL mRNA in HEPES buffer at a 1:3 ratio
(lipid mixture in ethanol, mRNA in HEPES buffer) using the
microfluidic device from Precision NanoSystems Inc (Vancouver BC,
Canada) at a 12 mL/minute flow rate. The mRNA/LNPs in 33% ethanol
were then incubated at room temperature for 60 minutes prior to
dialysis for 18 hours against 100 volumes (200 mL) of HEPES
buffer.
[0465] The polymer used for the sequential injection, polymer P1435
(NAG-C.sub.5N-PEG.sub.0.6k-[PEGMA300.sub.87.9%-PDSMA.sub.12.1%].sub.3.9kD-
a-b-[DMAEMA.sub.34.7%-BMA.sub.53.5%-PAA.sub.11.8%].sub.6.1kDa), was
solubilized at 20 mg/mL in HEPES buffer with agitation at 400 rpm
for 1 hour and then stored overnight at 4.degree. C. The polymer
was diluted to 7.5 mg/mL in HEPES buffer prior injection.
[0466] If mRNA/LNP and polymer were co-injected, a 2.times.
solution of each was prepared. Just prior to dosing, the solutions
were mixed and injected immediately.
[0467] The formulation particle size was measured by adding 10
.mu.L of formulation to 90 .mu.L of HEPES buffer into a disposable
micro-cuvette and analyzed using the Malvern Instrument ZETASIZER
NANO-ZS. The LNPs showed a particle size of 52 nm (z-average). The
formulation zeta-potential at pH 7.4 was measured by adding 10
.mu.L of formulation to 740 .mu.L of HEPES buffer into a disposable
1 mL cuvette. The formulation zeta-potential at pH 4 was measured
by adding 10 .mu.L of formulation to 740 .mu.L of sucrose acetate
buffer (pH 4) into a disposable 1 mL cuvette. The zeta dip cell was
inserted into the 1 mL cuvette and the formulation was analyzed
using the ZETASIZER NANO-ZS. Typically, the DOTMA LNPs had a zeta
potential of +12 mV at pH 7 and +16 mV at pH 4.0. The ability of
the LNP to compact the mRNA was measured in a 96 well plate using a
SYBR Gold dye accessibility assay. Typically, 50 .mu.L of the lipid
formulation at 0.01 mg/mL mRNA was added to 150 .mu.L of diluted
SYBR Gold stock solution (1 .mu.L of Stock SYBR Gold in 3 mL of
HEPES buffer) and incubated for 15 minutes at room temperature with
agitation (100 RPM). The fluorescence was read at an excitation
wavelength of 495 nm and emission wavelength of 538 nm. The percent
dye accessibility was calculated by dividing the fluorescence
intensity of the formulated mRNA by the fluorescence intensity of
the free mRNA.times.100. The DOTMA LNPs showed 2% dye accessibility
when prepared in HEPES buffer. Table 2 below shows a
characterization of an exemplary LNP formulation.
TABLE-US-00004 TABLE 2 Sample # RP450-2 Polymer or Lipid
DOTMA:CHEMS:CHOL:DMPE- PEG2K (50:32:13:5) N/P 27 Polymer or Lipid
Concentration 10.0 (mg/mL) Visual Appearance Opalescent (+) % Dye
Access HEPES pH 7.4 2% Z-Ave (nm) 52 PDI 0.200 Number (nm) 30 Pk 1
Mean Int (nm) 57 Pk 2 Mean Int (nm) 4191 Pk 1 Area Int (%) 97 Pk 2
Area Int (%) 4 ZP pH 7.4 (mV) 12 ZP pH 4 (mV) 16 Sizing data
quality Good
Example 2: In Vivo Expression of mRNA with Lipid-mRNA Formulations
and Co-Injection or Sequential Injection of Polymer
[0468] Female CD-1 mice (7-10 weeks old) were used for evaluating
the Flue mRNA/LNP+polymer formulations. The formulations were dosed
intravenously at 1 mg/kg of mRNA and 13 to 103 mg/kg of lipid, with
5 mice injected per group. Polymer P1435 alone at 75 mg/kg was
injected intravenously either as a co-injection or sequentially at
1, 5, 10, 30, 60 or 120 minutes post the Flue mRNA/LNP injection.
Mice injected with HEPES buffer was used as control. For each
injection mice were given a final dose volume of approximately 0.25
mL or 10 mL/kg based on individual body weights.
[0469] The in vivo expression of luciferase was evaluated by
detecting luminescence in mice using the Xenogen IVIS Lumina II
Imaging System (Caliper Life Sciences, now Perkin Elmer). The
imaging was performed at 6 hours following dosing. 15 minutes prior
to imaging, each mouse received 0.25 mL of D-luciferin (Perkin
Elmer), a luciferase substrate, at 15 mg/mL (dissolved in PBS) by
intra-peritoneal injection. A few minutes before imaging, mice were
place in an isoflurane chamber to induce anesthesia (isoflurane
concentration at .about.3%). Subsequently, mice were moved into the
IVIS imaging chamber, with the snout connected to an
isoflurane-filled nose cone with the mouse's ventral side up. The
luminescence images were acquired using Living Image software
(Caliper Life Sciences) with the exposure time, binning and F/Stop
remaining the same throughout the study. Mice were put back to the
cage as soon as the imaging was finished and they recovered within
1-3 minutes.
[0470] After the image acquisition was finished for all mice, the
luminescence results were analyzed using Living Image software.
Briefly, the color scale of each image was first adjusted to
display specific luminescence signal and eliminate background
signal. Then a region of interest (ROI) for the liver was defined
using the ROI tools, and ROI measure button was clicked to show the
photon flux data. Total flux (photons/sec) of the ROI on each
animal was used to represent the intensity of luminescence. Total
flux was averaged from all 5 mice for each formulation group for
comparison.
[0471] Table 3 displays luminescence values in the liver for
animals treated with DOTMA:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticle with or without sequential injection of polymer P1435
at 10 minutes following the first injection. Data was acquired at 6
hours post dose. Flue mRNA/LNP alone showed little luminescence
(only 3-fold above buffer) but with polymer P1435 sequential
injection, a 100-fold improvement in luminescence signal was
detected.
TABLE-US-00005 TABLE 3 Lipid mRNA Polymer Timing Total Flux
Lipid-mRNA Dose Dose Dose Between (photons/sec) Nanoparticle
(mg/kg) (mg/kg) Polymer (mg/kg) Injections Geomean STDEV Buffer 0 0
None 0 NA 3.38E+05 1.00E+00 DOTMA:CHEMS: 100 1 None 0 NA 6.24E+05
2.66E+05 CHOL:DMPE- 100 1 P1435 75 10 min 6.97E+07 4.86E+07 PEG2K
(50:32:13:5) N:P 27 + Fluc mRNA
[0472] Table 4 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticle with or without sequential injection of polymer P1435
or polymer P1299 at 10 minutes following the first injection. N:P
ratios from 14 to 27 and 2-5 mol % DMPE-PEG2k variations were
evaluated. Data was acquired at 6 hours post dose. Again the
mRNA/LNP alone showed little luminescence but with polymer P1435
sequential injection, a 100-fold improvement in luminescence signal
was detected. Reducing the N:P ratio from 27 to 14, and reducing
the DMPE-PEG2k from 5 to 3.5 mol % further improved the
luminescence signal by another 3-fold. Sequential injection of
polymer P1299
(NAG-C.sub.5N-PEG.sub.0.6k-[PEGMA300.sub.80%-PDSMA.sub.10%-BPAM.sub-
.10%].sub.3.5kDa-b-[DMAEMA.sub.34%-BPAM.sub.56%-PAA.sub.10%].sub.6.3kDa)
showed 5-fold improvement in luminescent signal compared to
mRNA/LNP alone.
TABLE-US-00006 TABLE 4 DMPE- Lipid mRNA Timing Total Flux
Lipid-mRNA PEG2k Dose Dose Between (photons/sec) Nanoparticle N:P
mol % (mg/kg) (mg/kg) Polymer Injections Geomean STDEV Buffer NA NA
0 0 None NA 2.58E+05 NA DOTAP:CHEMS: 27 5 113 1 None NA 1.70E+06
8.94E+05 CHOL:DMPE- 27 5 113 1 P1435 10 min 1.38E+08 1.88E+08 PEG2K
21 5 88 1 75 mg/kg 1.61E+08 9.48E+07 (2-5%) 14 5 59 1 2.51E+08
2.07E+08 (50:32:13:X 27 3.5 107 1 3.43E+08 9.68E+07 mol %) + 14 3.5
56 1 3.80E+08 1.26E+08 Fluc mRNA 27 2 102 1 2.26E+08 2.24E+08 27 5
113 1 P1299 8.34E+06 1.22E+07 75 mg/kg
[0473] Table 5 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticle with or without sequential injection of polymer P1435
at 10 minutes following the first injection or co-injection. N:P
ratios from 3.5 to 14 and were evaluated. Data was acquired at 6
hours post dose. Again the mRNA/LNP alone showed little
luminescence but with polymer P1435 sequential injection, a
300-fold improvement in luminescence signal was detected. Reducing
the N:P ratio from 14 to 7, and reducing the DMPE-PEG2k to 2 mol %
resulted in nearly a 500-fold improvement in luminescence signal
compared to mRNA/LNP alone. Further reducing the N:P ratio to 3.5
resulted in lower luminescence. Sequential injection of mRNA/LNP
and polymer P1435 showed slightly better luminescence signal
compared to co-injection.
TABLE-US-00007 TABLE 5 Lipid mRNA Timing Total Flux Lipid-mRNA Dose
Dose Between (photons/sec) Nanoparticle N:P (mg/kg) (mg/kg) Polymer
Injections Geomean STDEV Buffer NA 0 0 None NA 3.19E+05 NA
DOTAP:CHEMS: 14 53 1 None NA 1.07E+06 1.31E+05 CHOL:DMPE- 3.5 13 1
P1435 10 min 5.82E+07 5.61E+07 PEG2K 7 26 1 75 mg/kg 5.07E+08
6.21E+08 (50:32:14.5:2 14 53 1 3.58E+08 3.93E+08 mol %) + 14 53 1
co- 2.48E+08 3.69E+08 Fluc mRNA injection
[0474] Table 6 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticle with sequential injection of polymer P1435 from 1 to
120 minutes following the first injection or co-injection. Data was
acquired at 6 hours post dose. The luminescence signal was similar
between 1 and 10 minutes and dropped from 30 to 120 minutes.
Sequential injection of mRNA/LNP and polymer P1435 showed four-fold
higher luminescence signal compared to co-injection.
TABLE-US-00008 TABLE 6 Timing mRNA Total Flux Lipid-mRNA Between
Dose (photons/sec) Nanoparticle Polymer Injections (mg/kg) Geomean
STDEV Buffer None NA 0 2.52E+05 NA DOTAP:CHEMS: P1435 co-injection
1 1.57E+08 1.38E+08 CHOL:DMPE- 75 mg/kg 1 min 1 6.09E+08 3.40E+08
PEG2K 5 min 1 8.24E+07 2.28E+08 (50:32:16:2) 10 min 1 3.22E+08
2.43E+08 N:P 7 30 min 1 7.69E+07 5.23E+07 26 mg/kg + 60 min 1
1.57E+07 1.24E+07 Fluc mRNA 120 min 1 6.03E+06 1.30E+07
[0475] Table 7 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticles with sequential injection of polymer P1435 at 1
minute following the first injection. Data was acquired at 6 hours
post dose. In this study, two different Fluc mRNAs were tested.
Fluc 2 mRNA showed a 15-fold improvement in luminescence signal
compared to Fluc 1 mRNA. Fluc 2 mRNA contains Pseudo U only, a Cap
1 structure obtained from enzymatic capping and a longer poly A
tail (approximately double that of Fluc 1--.about.220 bases)
compared to Fluc 1 which has an ARCA cap structure, Pseudo
U/5-methyl-C modifications, and a poly A tail length of 120
bases.
TABLE-US-00009 TABLE 7 Timing mRNA Total Flux Lipid-mRNA Fluc
Between Dose (photons/sec) Nanoparticle Polymer mRNA Injections
(mg/kg) Geomean STDEV Buffer None None NA 0 1.82E+05 NA
DOTAP:CHEMS: P1435 Fluc 1 1 min 1 2.10E+08 1.57E+08 CHOL:DMPE- 75
mg/kg mRNA PEG2K Fluc 2 1 3.04E+09 2.12E+09 (50:32:16:2) mRNA N:P 7
26 mg/kg
Example 3: Synthesis of PEG.sub.0.6k-CTA (Compound 6)
##STR00015##
[0477] HOOC-PEG.sub.0.6K-ECT (Compound 6). To a 100 mL one-neck
round-bottom flask was added ECT (473 mg, 2.0 mmol, Omm Scientific)
followed by anhydrous tetrahydrofuran (20 mL) and triethylamine
(0.307 mL, 2.2 mmol). This mixture was stirred at 0.degree. C. for
5 min before trifluoroacetic acid pentafluorophenyl ester (0.368
mL, 2.14 mmol) was added drop wise to the stirred reaction. The
mixture was stirred at 0.degree. C. for 5 min then warmed to room
temperature.
[0478] After allowing to react for 20 min at room temperature, the
reaction was diluted into EtOAc (100 mL) and extracted with
saturated aqueous solution of NaHCO.sub.3 (3.times.40 mL). The
EtOAc layer was separated, dried over Na.sub.2SO.sub.4, filtered
and then evaporated providing the crude PFP-ester 4 as yellow
oil.
[0479] The crude ester 4 was dissolved in anhydrous
CH.sub.2Cl.sub.2 (20 mL) and then cooled to 0.degree. C. To the
cooled stirred solution was added triethylamine (0.251 mL, 1.8
mmol) and Amino-dPEG12-acid (1.12 g, 1.8 mmol, Quanta Biodesign),
and the mixture was warmed to room temperature. After stirring for
20 min at room temperature, the reaction mixture was evaporated
using a rotary evaporator providing yellow oil. The yellow oil was
dissolved in CH.sub.2Cl.sub.2 (approximately 2 mL) and the product
was purified by flash chromatography (SiO.sub.2, column size 5.0 cm
ID.times.10.0 cm length; isocratic elution with 100%
CH.sub.2Cl.sub.2 for 500 mL; then CH.sub.2Cl.sub.2/MeOH, 20:1 v/v
for 500 mL; then CH.sub.2Cl.sub.2/MeOH, 10:1 v/v for 3.0 L). The
product-containing fractions, as determined by TLC, were combined,
and the solvent was removed by rotary evaporation providing 750 mg
(48%) of the desired compound 6 as orange oil. H NMR (CD30D):
.delta. 1.35 (t, 3H, J=7.5 Hz, CH.sub.3), 1.89 (s, 3H, CH.sub.3),
2.38-2.57 (m, 6H), 3.32-3.41 (m, 4H), 3.50-3.75 (m, 48H).
Example 4: Synthesis of Na(OAc4)C5N-PEG.sub.0.6K-CTA (Compound
8)
Step 1. Synthesis of Compound 3
##STR00016##
[0481] N-t-Boc-5-amino-1-pentanol. To a 1.0 L one-neck round-bottom
flask containing a solution of 5-amino-1-pentanol (15.0 g, 145.4
mmol) in water (140 mL) and saturated aqueous NaHCO.sub.3 (1.4 mL),
a solution of di-tert-butyl dicarbonate (33.3 g, 152.7 mmol) in THF
(280 mL) was added. The mixture was then stirred at room
temperature overnight with the flask open to the atmosphere. The
reaction mixture was diluted with saturated aqueous NaHCO.sub.3 (90
mL) and extracted with EtOAc (400 mL). The organic layer was
separated, dried over Na.sub.2SO.sub.4, filtered, and the solvent
was evaporated providing 28.9 g (98%) of the final product as clear
colorless oil. .sup.1H NMR analysis showed the product was clean of
impurities, and no further purification was attempted.
Alternatively, N-t-Boc-5-amino-1-pentanol can be obtained from TCI
America of Portland, Oreg.
[0482] Compound 2. Compound 2 was prepared by a procedure adopted
from the literature (Westerlind, U. et al. Glycoconj. J. 2004, 21,
227-241). To a 500-mL one-neck round-bottom flask was added
2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-galactopyranose 1
(12.8 g, 32.8 mmol) followed by anhydrous CH.sub.2Cl.sub.2 (150 mL)
and trimethylsilyl trifluoromethanesulfonate (14.3 mL, 79.2 mmol).
This mixture was stirred at reflux overnight (ca. 18 h) under a
flow of argon gas. The reaction mixture was cooled to 0.degree. C.
and treated with triethylamine (6.4 mL, 45.9 mmol) for 30 min
before being warmed to room temperature, then washed with saturated
aqueous NaHCO.sub.3 (100 mL). The organic layer was separated and
dried over Na.sub.2SO.sub.4, filtered and evaporated providing
crude oxazoline intermediate. To the crude oxazoline product was
added anhydrous CH.sub.2Cl.sub.2 (200 mL),
N-t-Boc-5-amino-1-pentanol (10.0 g, 49.2 mmol) and 3 .ANG.
molecular sieves (18.0 g, dried at 150.degree. C. for >24 h).
This mixture was stirred at room temperature for 30 min under a
blanket of argon gas. Trimethylsilyl trifluoromethanesulfonate
(2.97 mL, 16.4 mmol) was added to the reaction mixture, and the
solution was stirred at room temperature overnight. The solution
was cooled to 0.degree. C. and treated with triethylamine (3.2 mL,
23.07 mmol) for 30 min before being warmed to room temperature.
After the reaction reached room temperature the mixture was
filtered, and the mother liquor was evaporated providing the crude
product as brown oil which was dissolved in anhydrous pyridine (100
mL) and treated with acetic anhydride (36 mL, 38.2 mmol). This
mixture was stirred under an argon atmosphere at room temperature
overnight, then evaporated under vacuum yielding a brown liquid,
which was dissolved in CH.sub.2Cl.sub.2 (200 mL). The solution was
vigorously stirred with a saturated aqueous NaHCO.sub.3 solution
(100 mL) and solid NaHCO.sub.3 in an open flask at room temperature
to quench remaining Ac.sub.2O and the organic layer was separated.
The aqueous layer was extracted with CH.sub.2Cl.sub.2 (1.times.200
mL) and all organic layers were combined. The organic layers were
washed with saturated aqueous NaHCO.sub.3 solution (1.times.100
mL), separated, dried over Na.sub.2SO.sub.4, filtered and
evaporated providing the crude product as a brown oil which was
then dissolved in CH.sub.2Cl.sub.2 (15 mL) and purified using
column chromatography (SiO.sub.2, column size 7.5 cm ID.times.16.0
cm length, EtOAc: Hexanes 1:3 v/v for 500 mL, EtOAc:Hexanes 4:1 v/v
for 500 mL, 100% EtOAc for 1.0 L, 10% MeOH in EtOAc v/v for 3.0 L).
Product-containing fractions were pooled and evaporated under
vacuum to a white solid which was further purified by trituration
with ether to yield the desired product as a white solid (5 g,
29%). ESI MS [M+H].sup.+ m/z 533.4.
[0483] Compound 3. To a 100 mL round bottom flask was added
Compound 2 (3.14 g, 5.9 mmol) followed by trifluoroacetic acid (10
mL, TFA). The mixture was stirred until all of the carbohydrate was
completely dissolved, then the TFA was evaporated under vacuum to
yield light yellow oil. To the oily residue was added diethyl ether
(10 mL), the mixture was sonicated for 2-5 min, and the supernatant
was decanted. The trituration process was repeated (3.times.10 mL
Et.sub.2O), and the crude product was dried under vacuum to yield a
white foam (3.2 g), which was used as described below.
Step 2
##STR00017##
[0485] Compound 7. To a 250 mL one-neck round-bottom flask was
added Compound 6 (3.37 g, 3.9 mmol, HPLC purified) followed by
anhydrous CH.sub.2Cl.sub.2 (40.0 mL), and triethylamine (2.17 mL,
15.6 mmol). This solution was stirred at 0.degree. C. under a low
flow of argon gas for 5 min before trifluoroacetic acid
pentafluorophenyl ester (737 .mu.L, 4.29 mmol) as added dropwise to
the reaction mixture. Then the mixture was warmed to room
temperature and was stirred at room temperature for 30 min.
[0486] The reaction progress was followed by TLC (SiO.sub.2,
CH.sub.2Cl.sub.2 and MeOH, 9:1 v/v) by looking for the
disappearance of the starting material (R.sub.f=0.30) and the
appearance of the PFP activated product (R.sub.f=0.64). Once the
starting material was consumed by TLC, the crude reaction was
diluted with CH.sub.2Cl.sub.2 (300 mL) and the mixture was
extracted using NaHCO.sub.3 (3.times.50 mL). The organic layer was
separated, dried over Na.sub.2SO.sub.4, filtered and evaporated
providing 3.9 g (97%) of the final product as orange oil. All
solvents and volatile reagents were thoroughly removed using high
vacuum overnight before the crude product is carried on to the next
synthetic step.
[0487] Compound 8. To a 100 mL one-neck round-bottom flask was
added Compound 7 (3.6 g, 3.5 mmol) followed by anhydrous
acetonitrile (7.5 mL) and triethylamine (1.46 mL, 10.5 mmol). The
mixture was stirred under a flow of argon gas until all of the
material was dissolved, then cooled to 0.degree. C. with an ice
bath. Deprotected amine 3 (1.81 g, 3.32 mmol) was dissolved in
anhydrous acetonitrile (7.5 mL), and the resulting solution was
added to the reaction mixture at 0.degree. C. dropwise over 5 min.
The reaction was allowed to warm to room temperature and was
stirred at room temperature overnight. The solvents were evaporated
using a rotary evaporator, and the crude product was dried under
high vacuum. The reaction progress was followed by analytical HPLC
by diluting the reaction mixture (5 .mu.L) into CH.sub.3CN (695
.mu.L) and 50 .mu.L of the diluted mixture was analyzed by HPLC
(10% CH.sub.3CN for 2 min, then linear gradient from 10% to 60%
CH.sub.3CN over 20 min, total flow rate of 1.0 mL/min). The desired
product had a retention time of 21.0 min.
[0488] The crude product was dissolved in MeOH (approximately 40
mL) and purified in 2-mL aliquots using preparative reverse phase
HPLC (Phenomenex, Luna 5C18(2), 100 .ANG., 25.0 cm.times.21.2 mm,
equipped with a SecurityGuard PREP Cartridge, C18 15.times.21.2 mm
ID, CH.sub.3CN/H2O, 30% CH.sub.3CN for 5 min, then linear gradient
from 30% to 53% CH.sub.3CN over 20 min, total flow rate of 20.0
mL/min,). The desired product eluted between 22.0 and 23.0 min. All
the fractions containing the desired product were combined, and the
solvent was completely removed using a rotary evaporator to yield
2.54 g (60%) of compound 8 after overnight drying under vacuum.
[0489] ESI MS: m/z 1277.6 ([M+H].sup.+1), 650.6 ([M+Na+H].sup.+2),
658.5 ([M+K+H].sup.+2), 661.7 ([M+2Na].sup.+2), 669.7
([M+Na+K].sup.+2), 677.5 ([M+2K].sup.+2).
[0490] 1H NMR (CD3OD): .delta. 1.35 (t, 3H, J=7.5 Hz), 1.33-1.62
(m, 6H), 1.88 (s, 3H), 1.93 (s, 3H), 1.95 (s, 3H), 2.03 (s, 3H),
2.15 (s, 3H), 2.32-2.56 (m, 6H), 3.15-3.25 (m, 2H), 3.25-3.42 (m,
6H), 3.50-3.70 (m, 44H), 3.97-4.20 (m, 4H), 4.55 (d, 1H, J=8.4 Hz),
5.05 (dd, 1H, J.sub.1=11.4 Hz, J.sub.2=3.4 Hz), 5.33 (dd, 1H,
J.sub.1=3.4 Hz, J.sub.2=0.9 Hz).
Example 5: Preparation of Na(OH)C5N-PEG.sub.0.6K-CTA (Compound
8a)
##STR00018##
[0492] Nag(OH)C5N-PEG.sub.0.6K-CTA (Compound 8a) was prepared in a
similar manner to the Nag(OAc4)C5N-PEG.sub.0.6K-CTA in Example 4
(Compound 8) except that compound 3 in Example 4 is replaced by the
unprotected sugar compound of compound 3a and the coupling reaction
between compound 6 of Example 3 and compound 3 of Example 4 has
been modified as shown below for compounds 6a and 3a.
[0493] Compound 3a is prepared as follows from compound 3b.
##STR00019##
[0494] To a 250 mL one-neck round-bottom flask was added compound
3b (1.86 g, 3.5 mmol) followed by 4M HCl in dioxane (30 mL). This
mixture was stirred and sonicated until all of the sugar was
completely dissolved. Then the mixture was evaporated on a rotary
evaporator providing an oily residue. To completely remove all HCl
gas the compound was dissolved in dioxane (30 mL) and solvents
removed by rotary evaporation. The solvent exchange process was
performed a total of 3 times to completely remove all HCl. Then the
flask was put under high vacuum for >30 min providing a white
foam solid. The crude compound was dissolved in anhydrous MeOH (25
mL) and treated with 0.5 M sodium methoxide solution in MeOH (5.80
g, 7.175 mL, 3.59 mmol, 1.025 eq, measured by weight to ensure
accuracy of addition). The first equivalent of NaOMe is used to
de-protonate the quaternary amine salt liberating the free amine.
Only a slight excess of NaOMe beyond one equivalent (i.e., 0.025
eq, 0.09 mmol) is needed to facilitate the acetyl deprotection.
Once NaOMe is added the mixture is then stirred under a flow of
argon overnight at room temperature. Reaction progress was
monitored by LCMS using Agilent Q-TOF Liquid Chromatography Mass
Spectrometer by dissolving the product in MeOH at ca. 1.0 .mu.g/mL.
The LC used a C18 UPLC column (Agilent Eclipse Plus C18, catalog
number 959757-902, 1.8 m, 2.1 mm.times.50 mm, column at room
temperature, CH.sub.3CN/H.sub.2O containing 0.1% formic acid,
isocratic gradient at 5% CH.sub.3CN for 1 min, then linear gradient
from 5% to 90% CH.sub.3CN over 4 min, total flow rate of 0.4
mL/min). The desired product elutes between 0.4-0.5 min using the
above HPLC conditions while the crude intermediate product (i.e.,
Boc removed with acetyls still present) elutes between 2.0-2.2 min.
Once the sugar was fully de-protected the catalytic NaOMe (0.09
mmol) is quenched by adding a slight excess of acetic acid (10
.mu.L, 0.175 mmol) to the reaction mixture. Then all solvents are
removed by evaporating on a rotary evaporator. This process yielded
1.1 g (100%) of the final product as a white solid. The final
product was characterized using a 400 MHz 1H NMR with CD.sub.3OD as
solvent and all spectra were consistent with the desired product
compound 3a.
[0495] Nag(OH)C5N-PEG.sub.0.6K-CTA (Nag(OH)C5N-PEG.sub.12-CTA;
Compound 8a) was prepared as follows. Compound 6a was prepared as
in Example 3 (Compound 6).
[0496] To a 250 mL one-neck round-bottom flask was added compound
6a (3.17 g, 3.68 mmol) followed by anhydrous acetonitrile (10 mL).
In a separate flasks the compound 3a (1.07 g, 3.5 mmol) was
dissolved in anhydrous DMF (10 mL). Once compound 3a was partially
dissolved as a milky white suspension the solution was transferred
to a 100 mL addition funnel. In another flask was added PyBOP (2.0
g, 3.85 mmol) and anhydrous DMF (10 mL). The PyBOP/DMF solution was
taken up into a 20 mL syringe. Then all 3 solutions (compound
6a/CH.sub.3CN, compound 3a/DMF, and PyBOB/DMF) were combined
simultaneously and as fast as possible while the reaction solution
was vigorously stirred. Once the additions were complete the
reaction was treated with N,N-diisopropylethylamine (1.22 mL, 7.0
mmol) and the solution was stirred at room temperature under a flow
of argon gas for 30 min. The reaction progress was determined using
Agilent Q-TOF Liquid Chromatography Mass Spectrometer by dissolving
the crude reaction (1.0 .mu.L) into MeOH (1.0 mL) and injecting 1.0
.mu.L (FIGS. 1-2). The LC used a C18 UPLC column (Agilent Eclipse
Plus C18, catalog number 959757-902, 1.8 .mu.m, 2.1 mm.times.50 mm,
column at room temperature, CH.sub.3CN/H.sub.2O containing 0.1%
formic acid, isocratic gradient at 5% CH.sub.3CN for 1 min, then
linear gradient from 5% to 90% CH.sub.3CN over 4 min, total flow
rate of 0.4 mL/min). The desired product elutes between 3.0-3.1 min
using the above HPLC conditions. The sugar starting material (i.e.,
compound 3a) was not detected on the mass spec analysis after the
reaction was stirred at room temperature for 30 min. Mass spec
analysis confirms the presence of compound 8a
[M+Na].sup.+1=1173.5207 m/z; [M+H].sup.+1=1151.5397 m/z).
[0497] After reacting for 30 min the crude reaction mixture of
compound 8a was diluted by the addition of H.sub.2O (25 mL) and
purified using C18 preparative reverse phase HPLC by Shimadzu
(Phenomenex, Luna 5C18(2), part number 00G-4252-P0-AX, 100 .ANG.,
25.0 cm.times.21.2 mm, with a SecurityGuard PREP Cartridge, C18
15.times.21.2 mm ID, part number AJ0-7839, CH.sub.3CN/H2O with
0.01% TFA, isocratic gradient at 5% CH.sub.3CN for 5 min, then
linear gradient from 5% to 50% CH.sub.3CN over 17 min, then 50% to
53% CH3CN over 3 min, total flow rate of 20.0 mL/min, column at
room temperature). 2.0 mL of the crude compound dissolved in
DMF/H.sub.2O (ca. 75 mg/mL) were injected each HPLC run. Using the
HPLC purification conditions above the desired product compound 8a
eluted between 21.5 and 22.5 min. All the fractions containing the
desired product were combined and the water/CH3CN solvent was
completely removed using a rotary evaporator then high vacuum
overnight. The combined yield of the final product after HPLC
purification and overnight high vacuum produced 3.05 g (76%) of the
desired product as a bright orange solid. .sup.1H NMR analysis was
consistent with the presence of the desired product compound
8a.
##STR00020##
Example 6: General Procedure for Polymer Synthesis
[0498] First block synthesis general procedure: The first block
polymer is prepared using the following approximate ratios:
[Monomer/CTA/Initiator]=[15-20/1/0.5] at approximately 1.3 M in
DMF. Following oxygen purge with Nitrogen or Argon, the
polymerization reaction is heated to 60-68.degree. C. for a
particular amount of time (generally 1 h 15 min-3 h) until the
desired molecular weight is reached. The polymerization reaction is
stopped by placing in an ice bath and opening the reaction to air.
The desired polymer is purified by dialysis against methanol (3-7
days) using 2 KDa MWCO dialysis tubing. The resulting polymer is
isolated by removing solvent under reduced atmosphere.
[0499] Second block synthesis general procedure: The second block
polymer is prepared using the following approximate
ratios::[Monomer/CTA/Initiator]=[100-130/1/0.5] at approximately
2-3 M in DMF. Following oxygen purge with Nitrogen or Argon, the
polymerization reaction is heated to 60-68.degree. C. for a
particular amount of time (generally 3-6 h) until the desired
molecular weight is reached. The polymerization reaction is stopped
by placing in an ice bath and opening the reaction to air. The
desired polymer is purified by precipitation into
diethylether/hexanes and/or dialysis against methanol (3-5 days)
using 2 KDa MWCO dialysis tubing. The resulting polymer can be
isolated by removing solvent under reduced atmosphere, or dialysis
against water using 2 KDa MWCO dialysis tubing, followed by
lyophilization.
Example 7: Determining Monomer Incorporation within Individual
Blocks of a Polymer During Polymer Synthesis
[0500] The amount of a given monomer within a given polymer block,
typically the first or hydrophilic polymer block, of the polymers
exemplified and claimed herein has been determined by the following
procedure. Samples taken before and after the polymerization
reaction (i.e., T.sub.0 (time zero) and T.sub.f (time final)) are
analyzed by analytical HPLC to determine the extent of monomer
consumption and/or monomer incorporation.
[0501] The initial monomer amounts in the polymerization reaction
(time 0, T.sub.0) are determined by sampling the polymerization
reaction solution prior to nitrogen or argon purge. A (20 .mu.L)
sample of the reaction solution is withdrawn from the reaction
solution and diluted into 180 .mu.L of Methanol (MeOH). A portion
of the resulting solution (10 .mu.L) is further diluted into 590
.mu.L MeOH, to afford a test sample with an overall dilution of
1:600 (from the polymerization reaction) for analysis by analytical
HPLC.
[0502] Upon completion of the polymerization reaction a time final
(T.sub.f) sample is prepared analogous to the T.sub.0 sample
described above.
[0503] Analytical HPLC analysis of the T.sub.0 and T.sub.f samples
are performed using a C18 Phenomenex 5.mu. 100 .ANG. 250.times.4.6
mm.times.5 micron (Part#00G-4252-E0) Luna column with guard column
heated to 30.degree. C. Three independent dilutions for each time
point (i.e., T.sub.0, and T.sub.f) are prepared and analyzed for
each time point. A 10 .mu.l of sample is injected onto the column
and eluted with the following gradient. Hold an isocratic eluent of
5% acetonitrile/water with 0.1% TFA for 2 minutes. Switch to a
linear gradient from 5% to 95% acetonitrile over 25 minutes. Hold
an isocratic eluent of 95% acetonitrile for 5 minutes. Return to 5%
acetonitrile over 0.01 minutes. Hold the isocratic eluent of 5%
acetonitrile/water with 0.1% TFA for 5 minutes. At least three
independent sample preparations for both T.sub.0 and T.sub.f were
used for the calculation of monomer incorporation within the
block.
[0504] The following methodology is used to calculate the %
incorporation of a given monomer: [0505] a. Calculate the average
T.sub.0, and T.sub.f monomer peak areas from the three independent
sample preparations [0506] b. Calculate the consumption of
individual monomers in the reaction (monomer % consumption): [0507]
=(1-(T.sub.f-avg monomer peak area/T.sub.0-avg monomer peak
area).times.100. [0508] c. Calculate the molar fraction consumed of
the individual monomers based on monomer input percent [0509]
=(Monomer % conversion (calculated in step (b)
above).times.0.01).times.monomer feed %. [0510] d. Total monomer
consumption in the polymerization reaction and overall percent
conversion: [0511] i. Total monomer consumption=sum of molar
fraction consumed for the individual monomers calculated in step
(c) above. [0512] ii. Overall % conversion=Total monomer
consumption (calculated in step (d)(i) above).times.100. [0513] e.
Calculate the percent monomer incorporation for each monomer in the
polymer [0514] i. =(Monomer molar fraction consumed (step (c)
above)/total monomer consumed (step (d)(i) above).times.100.
Example 8: Determining Monomer Incorporation within Individual
Blocks of a Polymer During Polymer Synthesis
[0515] The amount of a given monomer within a given polymer block,
typically the second polymer block or the polymer block containing
PAA, BMA and DMAEAMA, of the polymers exemplified and claimed
herein has been determined by the following procedure. Samples
taken before and after the polymerization reaction (i.e., T.sub.0
(time zero) and T.sub.f(time final)) are analyzed by analytical
HPLC to determine the extent of monomer consumption and/or monomer
incorporation.
[0516] The initial monomer amounts in the polymerization reaction
(time 0, T.sub.0) are determined by sampling the polymerization
reaction solution prior to nitrogen purge. A (20 .mu.L) sample of
the reaction solution is withdrawn and diluted into 180 .mu.L of
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/Methanol (MeOH)/Nano-pure
water (H.sub.2O) (2:1:1, v/v) containing 0.1% TFA. A portion of the
resulting solution (10 .mu.L) is further diluted into 590 .mu.L of
HFIP/MeOH/H.sub.2O (2:1:1, v/v) containing 0.1% TFA, to afford a
test sample with an overall dilution of 1:600 (from the
polymerization reaction) for analysis by analytical HPLC.
[0517] Upon completion of the polymerization reaction a time final
(T.sub.f) sample is prepared analogous to the T.sub.0 sample
described above. A (20 .mu.L) sample of the reaction solution is
withdrawn and diluted into 180 .mu.L of
1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/Methanol (MeOH)/Nano-pure
water (H.sub.2O) (2:1:1, v/v) containing 0.1% TFA. A portion of the
resulting solution (10 .mu.L) is further diluted into 590 .mu.L of
HFIP/MeOH/H.sub.2O (2:1:1, v/v) containing 0.1% TFA, to afford a
test sample with an overall dilution of 1:600 (from the
polymerization reaction) for analysis by analytical HPLC.
[0518] Analytical HPLC analysis of the T.sub.0, and T.sub.f samples
are performed using a C18 Phenomenex 5.mu. 100 .ANG. 250.times.4.6
mm.times.5 micron (Part#00G-4252-E0) Luna column with guard column
heated to 30.degree. C. Three independent dilutions for each time
point (i.e., To, and T.sub.f) are to be prepared and analyzed. A 10
.mu.l of sample is injected onto the column and eluted with the
following gradient. Hold an isocratic eluent of 5%
acetonitrile/water with 0.1% TFA for 10 minutes. Switch to a linear
gradient from 5% to 15% acetonitrile over 10 minutes. Switch to a
linear gradient from 15% to 95% acetonitrile over 20 minutes. Hold
an isocratic eluent of 95% eluent acetonitrile for 5 minutes.
Return to 5% acetonitrile over 0.01 minutes. Hold the isocratic
eluent of 5% acetonitrile/water with 0.1% TFA for 5 minutes. At
least three independent sample preparations for both T.sub.0, and
T.sub.f were used for the calculation of monomer incorporation
within the block.
[0519] The following methodology is used to calculate the %
incorporation of a given monomer: [0520] a. Calculate the average
T.sub.0, and T.sub.f monomer peak areas from the three independent
sample preparations [0521] b. Calculate the consumption of
individual monomers in the reaction (monomer % consumption): [0522]
=(1-(T.sub.f-avg monomer peak area/T.sub.0-avg monomer peak
area).times.100 [0523] c. Calculate the molar fraction consumed of
the individual monomers based on monomer input percent [0524]
=(Monomer % conversion (calculated in step
b).times.0.01).times.monomer feed % (for example, DMAEMA=0.25,
PAA=0.25, BMA=0.50) [0525] d. Total monomer consumption in the
polymerization reaction and overall percent conversion: [0526] i.
Total monomer consumption=sum of molar fraction consumed for the
individual monomers calculated in (c). [0527] ii. Overall %
conversion=Total monomer consumption (calculated in
(d)(i).times.100 [0528] e. Calculate the percent monomer
incorporation for each monomer in the polymer [0529] i. =(Monomer
molar fraction consumed (calculated in (c) above)/total monomer
consumed (calculated in (d)(i))).times.100
Example 9: Synthesis of Polymer
NagC5N-PEG.sub.0.6-[PEGMA4-5.sub.80-PDSMA.sub.10-BPAM.sub.10].sub.6.4-b-[-
D.sub.25-B.sub.50-P.sub.25].sub.6.3 (P1)
Example 9.1: Synthesis of Macro-CTA C1
##STR00021##
[0531] PEGMA4-5 (0.675 g, 2.25 mmol), PDSMA (0.072 g, 0.282 mmol),
BPAM (0.077 g, 0.282 mmol), Nag(OAc4)C5N-PEG.sub.0.6K-CTA (Compound
8) (0.090 g, 0.0704 mmol; 1:40CTA: Monomers), AIBN (0.578 mg,
0.00252 mmol; CTA:AIBN 20:1) and DMF (1.65 g) were introduced under
nitrogen in a sealed vial. The mixture was degassed by bubbling
nitrogen for 30 minutes, and the reaction was allowed to proceed at
68.degree. C. with rapid stirring for 2 hours. The reaction was
stopped by placing the vial in ice and exposing the mixture to air.
The polymer was purified by dialysis against methanol for 24 hours
(Spectrum Labs, Spectra/Por Dialysis Membrane MWCO: 2000), followed
by removal of solvents under vacuum. The resulting Macro-CTA was
dried under vacuum for 6 hours. The structure and composition of
the purified polymer were verified by .sup.1H NMR, which also
confirmed the absence of signals corresponding to vinyl groups of
un-incorporated monomers. Purity of the polymer was confirmed by
GPC analysis. M.sub.n,GPC=7.7 kDa, dn/dc=0.05700, PDI=1.28.
Example 9.2: Synthesis of Polymer P1
##STR00022##
[0533] BMA (0.246 g, 1.73 mmol), PAA (0.099 g, 0.87 mmol), DMAEMA
(0.136 g, 0.87 mmol), MacroCTA C1 (0.113 g, 0.0147 mmol;
1:236CTA:Monomers), AIBN (0.241 mg, 0.00147 mmol; CTA:AIBN 10:1)
and DMF (0.615 g) were introduced in a vial. The mixture was
degassed by bubbling nitrogen into the mixture for 30 minutes, and
then allowed to react for 10 hr at 67-68.degree. C. The reaction
was stopped by placing the vial in ice and exposing the mixture to
air. The polymer was purified by dialysis from acetone/DMF 1:1 into
hexane/ether 75/25 (three times). The resulting polymer was dried
under vacuum for at least 8 hours. The structure and composition of
the purified polymer were verified by .sup.1H NMR, which also
confirmed the absence of signals corresponding to vinyl groups from
un-incorporated monomers. GPC analysis: M.sub.n=13.996 kDa,
dn/dc=0.056505, PDI=1.26.
[0534] The acetyl groups were removed by treatment of the polymer
with sodium methoxide (6 equivalents) in anhydrous
methanol/chloroform under an atmosphere of argon at room
temperature for 1.0 hour. The polymer was capped with
2,2'-dipyridyl disulfide (2 equivalents relative to pyridyl
disulfide residues in the polymer) at room temperature for 1.0 hour
under a flow of argon gas. After the capping the reaction was
diluted with MeOH and filtered. The filtrate was transferred to a
dialysis membrane with a 2000 g/mol molecular weight cut off
(Spectrum Labs, Spectra/Por Dialysis Membrane MWCO: 2000) and
dialyzed against MeOH over 24 hours followed by dialysis against
water. The solvent was evaporated, and the polymer was dried under
vacuum.
Example 10: Synthesis of Polymer
NagC5N-PEG.sub.0.6-[PEGMA4-5.sub.80-PDSMA.sub.10-BPAM.sub.10].sub.7.2-b-[-
D.sub.25-B.sub.50-P.sub.25].sub.6.1 (P2)
Example 10.1: Preparation of MacroCTA C2
##STR00023##
[0536] MacroCTA C2 was prepared as described in Example 9.1
starting from PEGMA4-5 (8.083 g, 27.0 mmol), PDSMA (0.860 g, 3.37
mmol), BPAM (0.921 g, 3.37 mmol), Nag(OAc4)C5N-PEG.sub.0.6K-CTA
(Compound 8) (1.076 g, 0.842 mmol; 1:40CTA:Monomers), AIBN (6.914
mg, 0.0421 mmol; CTA:AIBN 20:1) and DMF (19.73 g). Polymerization
time was 2 hr 55 min. GPC: M.sub.n=8.500 kDa; PDI.about.1.23;
dn/dc=0.5780.
Example 10.2: Preparation of Polymer P2
##STR00024##
[0538] Extension of MacroCTA C2 by RAFT polymerization was carried
out as described in Example 10.1 using BMA (0.553 g, 3.89 mmol),
PAA (0.226 g, 1.98 mmol), DMAEMA (0.311 g, 1.98 mmol), MacroCTA C2
(0.560 g, 0.0659 mmol; 1:118CTA:Monomers), AIBN (1.082 mg, 0.00659
mmol; CTA:AIBN 10:1) and DMF (1.37 g+0.69 g). Polymerization was
stopped after 5 hours, and the product was purified by dialysis
from Acetone/DMF 1:1 into hexane/ether 75/25 (three times). GPC:
dn/dc=0.053188; M.sub.n=14.7 kDa; PDI=1.31. The acetyl groups were
removed with NaOMe as described in Example 9.2.
Example 11: Synthesis of Polymer
NagC5N-PEG.sub.0.6-[PEGMA4-5.sub.80-PDSMA.sub.10-BPAM.sub.10].sub.7.2-b-[-
D.sub.25-B.sub.50-P.sub.25].sub.10.8 (P3)
##STR00025##
[0540] MacroCTA C2 (Example 10) was extended by RAFT polymerization
as described in Example 10.2 using BMA (0.197 g, 1.39 mmol), PAA
(0.079 g, 0.69 mmol), DMAEMA (0.109 g, 0.69 mmol), Macro-CTA (0.100
g, 0.0118 mmol; 1:236CTA:Monomers), AIBN (0.193 mg, 0.00118 mmol;
CTA:AIBN 10:1) and DMF (0.492 g) for 4.5 hours, and the product was
purified by dialysis from Acetone/DMF 1:1 into hexane/ether 75/25
(three times). GPC: dn/dc=0.053160; Mn=19.3 kDa; PDI=1.39. The
acetyl groups were removed with NaOMe as described in Example
10.2.
Example 12: Synthesis of Polymer
PEG.sub.0.6-[PEGMA4-5.sub.80-PDSMA.sub.10-BPAM.sub.10].sub.6.7-b-[D.sub.2-
5-B.sub.50-P.sub.25].sub.6.2 (P4)
Example 12.1: Preparation of MacroCTA C4
##STR00026##
[0542] Macro-CTA C4 was prepared as described in Example 9 starting
with PEGMA4-5 (5.128 g, 17.1 mmol), PDSMA (0.546 g, 2.14 mmol),
BPAM (0.584 g, 2.14 mmol), PEG.sub.0.6K-CTA (Compound 6) (0.461 g,
0.534 mmol; 1:40CTA:Monomers), AIBN (4.385 mg, 0.0267 mmol; CTA:
AIBN 20:1) and DMF (12.52 g); reaction time was 1 hr 40 min. GPC:
Mn=7.50 kDa; PDI.about.1.20; dn/dc=0.053910.
Example 12.2: Preparation of Polymer P4
##STR00027##
[0544] Synthesis and purification of Polymer P4 was carried out as
described in Example 8.2 using BMA (1.656 g, 11.6 mmol), PAA (0.676
g, 5.92 mmol), DMAEMA (0.931 g, 5.92 mmol), MacroCTA C4(1.5 g,
0.197 mmol; 1:118CTA:Monomers), AIBN (3.241 mg, 0.0197 mmol;
CTA:AIBN 10:1) and DMF (4.16 g+2.08 g). GPC: dn/dc=0.050;
M.sub.n=13.8 kDa; PDI=1.1.
Example 13: Synthesis of Polymer
NagC5N-PEG.sub.0.6-[PEGMA4-5.sub.80-PDSMA.sub.10-BPAM.sub.10].sub.6.6-b-[-
D.sub.25-B.sub.50-P.sub.25].sub.14.7 (P5)
Example 13.1: Preparation of MacroCTA C5
##STR00028##
[0546] MacroCTA C5 was synthesized as described in Example 9.1
starting from PEGMA4-5 (0.5 g, 1.67 mmol), PDSMA (0.053 g, 0.208
mmol), BPAM (0.057 g, 0.208 mmol), Nag(OAc4)C5N-PEG.sub.0.6K-CTA
(Compound 8) (0.0665 g, 0.0521 mmol; 1:40CTA:Monomers), AIBN (0.428
mg, 0.0026 mmol; CTA:AIBN 20:1) and DMF (1.22 g). Polymerization
time was 2 hr 30 min. GPC: Mn=7.85 kDa; PDI=1.18; dn/dc=0.066.
Example 13.2: Preparation of Polymer P5
##STR00029##
[0548] Synthesis and purification of Polymer P5 was carried out as
described in Example 9.2 using BMA (0.62 g, 4.36 mmol), PAA (0.249
g, 2.18 mmol), DMAEMA (0.342 g, 2.18 mmol), MacroCTA C5 (0.189 g,
0.0242 mmol; 1:360CTA:Monomers), AIBN (0.398 mg, 0.00242 mmol;
CTA:AIBN 10:1) and DMF (1.55 g). Polymerization was allowed to
proceed for 10 hrs. GPC: dn/dc=0.063851; M.sub.n=22.5 kDa;
PDI=1.41. Deprotection was carried out as described in Example
9.2.
Example 14: Synthesis of Polymer
NagC5N-PEG.sub.0.6-[PEGMA4-5.sub.80-PDSMA.sub.10-BPAM.sub.10].sub.3.5-b-[-
D.sub.25-B.sub.50-P.sub.25].sub.6.3 (P6)
Example 14.1: Preparation of MacroCTA C6
##STR00030##
[0550] Macro-CTA C6 was synthesized as described in Example 9.1
starting from PEGMA4-5 (1.503 g, 5.00 mmol), PDSMA (0.160 g, 0.626
mmol), BPAM (0.171 g, 0.626 mmol), Nag(OAc4)C5N-PEG.sub.0.6K-CTA
(Compound 8) (0.500 g, 0.391 mmol; 1:40CTA:Monomers), AIBN (3.213
mg, 0.0196 mmol; CTA:AIBN 20:1) and DMF (3.668 g); reaction time
was 1 hr 45 min. GPC: M.sub.n=4.8 kDa; PDI=1.19;
dn/dc=0.061481.
Example 14.2: Preparation of Polymer P6
##STR00031##
[0552] Synthesis and purification of Polymer P6 was carried out as
described in Example 9.2 using BMA (0.218 g, 1.54 mmol), PAA (0.089
g, 0.781 mmol), DMAEMA (0.123 g, 0.781 mmol), MacroCTA C6 (0.125 g,
0.0260 mmol; 1:118CTA:Monomers), AIBN (0.428 mg, 0.00260 mmol;
CTA:AIBN 10:1) and DMF (0.830 g). Polymerization was allowed to
proceed for 4 hrs and 50 min. GPC: dn/dc=0.05812; M.sub.n=11.1 kDa;
PDI=1.38. Deprotection was carried out as described in Example
9.2.
Example 15: Synthesis of Polymer
NagC5N-PEG.sub.0.6-[PEGMA4-5.sub.86-PDSMA.sub.14].sub.3.82KDa-[BMA.sub.45-
-PAA.sub.15-DMAEMA.sub.40].sub.5.98KDa (P7)
Example 15.1: Preparation of MacroCTA C7
##STR00032##
[0554] AIBN/DMF (21.93 g of 1.05603 mg/g ABIN in DMF) was added to
Nag(OH)C5N-PEG.sub.0.6K-CTA (synthesized as described in Example 5
compound 8a) (3.075 g; 2.6705 mmol) in a 40 ml reaction vessel and
mixed to dissolve the CTA. DMF was then added until the total
weight of DMF was 24.9627 g. To the resulting solution was added
PEGMA (11.18 g, 37.2621 mmol, filtered through aluminum oxide
(activated, basic, Brockmann I) and PDSMA (1.1211 g, 4.1393 mmol).
The resulting solution was mixed and then transferred to a sealed
50 mL round bottom flask equipped with a magnetic stir bar. The
resulting solution was de-oxygenated by bubbling nitrogen into the
solution for 50 min on ice. The flask was moved to room temperature
for 4 min and then placed in an oil bath pre-heated to 68.degree.
C. for 1 hour 42 minutes (stir speed was set at 350 rpm). The
reaction was stopped by placing the vial in ice and exposing the
mixture to air. The reaction solution was diluted with MeOH,
transferred to dialysis membranes (Spectrum Labs, Spectrum
Spectra/Por 6 Dialysis Membrane Tubing MWCO: 2000) and dialyzed
against MeOH (6.times.4000 mL) for 6 days. Samples were taken for
LC-MS, GPC and .sup.1H NMR analyses. After dialysis, the solvent
was removed under reduced atmosphere followed by high vacuum to
afford 2.45 g of polymer. LC-MS analysis indicated no residual CTA
peak. .sup.1H NMR, which also confirmed the absence of signals
corresponding to vinyl groups of un-incorporated monomers. Purity
of the polymer was confirmed by GPC analysis. M.sub.n,GPC=4.97 KDa,
PDI=1.12, dn/dc=0.06469, PDI=1.12.
Example 15.2: Synthesis of Polymer P7
##STR00033##
[0556] AIBN/DMF solution (7.0225 g; 1.10468 mg/g AIBN in DMF) was
added to macro-CTA C7 (2.350 g) in a 40 mL reaction vessel; the
sample was mixed to dissolve the macro-CTA. DMF was then added
until the total weight of DMF was 15.05 g. BMA (3.967 g, filtered
through aluminum oxide (activated, basic, Brockmann I), PAA (1.6217
g) and DMAEMA (2.237 g, filtered through aluminum oxide [activated,
basic, Brockmann I]) were added to the resulting solution and the
solution was mixed. The mixture was vortexed for several minutes to
give a homogeneous stock solution and transferred to a sealed 50 mL
round bottom flask equipped with a magnetic stir bar. The mixture
was then cooled to 0.degree. C. using an ice bath and maintained at
0.degree. C. while degassed by vigorously bubbling nitrogen inside
the solution for 55 minutes. The flask septa was placed into an oil
bath pre-heated to 61.degree. C. (stirring speed was 350) and
allowed to stir for 4 hours 30 minutes. The reaction was stopped by
placing the vial in ice and exposing the mixture to air. The
reaction was then diluted with acetone (roughly the same volume of
acetone as the DMF used in the reaction vial) and precipitated into
a stirred mixture of ether/hexanes (1:3 v/v) in a 50 mL centrifuge
tube once and then into a large beaker with 600 mL ether/hexanes
(1:3 v/v). The polymer precipitate was isolated and dissolved with
MeOH, transferred to three individual dialysis membranes (Spectrum
Labs, Spectrum Spectra/Por 6 Dialysis Membrane Tubing MWCO: 2,000)
and dialyzed against methanol (5.times.4000 mL) for 4 days. After
the dialysis against methanol, it was dialyzed against nanopure
water using the same membrane (.times.6, water changed every hour).
When the dialysis was complete, the solution was transferred to
tared vials and treated with liquid nitrogen before being
lyophilized for 5 days to afford 3.46 g of the final product. The
final product was analyzed by UV/vis, NMR, GPC and HPLC equipped
with RI detector (for batch dn/dc). Analysis of the polymer by
.sup.1H-NMR indicated a polymer with no vinyl groups remaining and
the presence of PDSMA. The NMR is consistent for proposed
structure. GPC results: Mn=10.936 KDa, PDI=1.30,
dn/dc=0.057867.
Example 16: Synthesis of Polymer
NAG-PEG.sub.0.6-[PEGMA.sub.100].sub.3.5k-[BMA.sub.49-PAA.sub.10-DMAEMA.su-
b.33-PDSMA.sub.8].sub.7.1k (P8)
Example 16.1: Preparation of MacroCTA C8
##STR00034##
[0558] To a 20 mL reaction vial was added to
Nag(OH)C5N-PEG.sub.0.6K-CTA (synthesized as described in Example 5,
compound 8a) (794.6 mg, 0.6922 mmol, CTA) followed by a solution of
AIBN (5.0438 g solution dissolved in DMF at a concentration of
1.1268 mg/g, 5.68 mg AIBN, 0.03461 mmol,
2,2'-azobis(2-methylpropionitrile), compound recrystallized from
MeOH) then an additional amount of DMF (432.2 mg) was added
bringing the total amount of DMF used in this reaction to 5.4760 g.
This solution was mixed and vortexed for several minutes until all
of the CTA was completely dissolved. Once all the CTA was
completely dissolved PEGMA (3219.3 mg, 10.730 mmol, poly(ethylene
glycol) methyl ether methacrylate with average M, =300 g/mol,
inhibited with 100 ppm MEHQ and 300 ppm of BHT inhibitors, Aldrich
part number 447935-500 mL, inhibitors removed by passing the neat
monomer through a plug of Al.sub.2O.sub.3, was added to the
reaction vial. This mixture was stirred for several minutes. The
reaction vial was partially sealed and cooled to 0.degree. C. using
an ice bath while the mixture was degassed by vigorously bubbling
nitrogen for 30 minutes with magnetic stirring of the reaction
solution. Then the vial was completely sealed and placed into a
heater block. The stirring speed was set at 300 rpm, the
thermometer was set at 68.degree. C. and was maintained at this
temperature during the entire process. The reaction was left to
stir at 68.degree. C. for 1 hours and 47 minutes. After the
reaction is complete it was quenched by opening the vial and then
placing the reaction vial in ice exposing the mixture to air. The
reaction vial was diluted with MeOH (10 mL) and transferred to a
dialysis membrane with a 2000 g/mol molecular weight cut off
(Spectrum Labs, Spectrum Spectra/Por 6 Dialysis Membrane Tubing
MWCO: 2000) and dialyzed against MeOH (3.times.4000 mL) for 4 days.
The dialysis solution was changed every day for 3 iterations total.
The polymer in the dialysis bag was analyzed according to the
following procedure: A small aliquot of the dialysis solution (ca.
500-1000 .mu.L) was withdrawn from the dialysis tubing and placed
into a tared vial. The solution was then evaporated using a rotary
evaporator. Once the solvents are removed the vial was transferred
to a high vacuum line and placed under high vacuum. The compound is
dried for <15 min. Once the vial weight is constant then the
compound was dissolved immediately in DMF with 1% weight LiBr
solution. The final concentration of the polymer was approximately
8 mg/mL in DMF with 1% wt LiBr (DMF measured by weight then
converted to volume). A 20 kDa polystyrene standard (Fluka, part
number 81407-1G) dissolved in DMF with 1% wt LiBr at a
concentration of roughly 3 mg/mL (DMF measured by weight then
converted to volume) is then injected (100 .mu.L) on the GPC
followed by the polymer sample of interest (60, 80, 100, and 120
.mu.L). Once the final GPC analysis is determined then the dialysis
solution was transferred to a 40 mL reaction vial then the solvents
were removed using a rotary evaporator. Then the material was place
on a high vacuum line (pressure <0.5 torr) for >24 hours.
This process provided 682.9 mg of the final product. The final
product is then analyzed by NMR and GPC. The final product was
stored at room temperature under high vacuum. The NMR is consistent
for proposed structure. GPC results: Mn=4.600, dn/dc=0.053354.
Example 16.2. Synthesis of Polymer P8
##STR00035##
[0560] To a 40 mL reaction vial was added macro-CTA C8 (682.1 mg,
0.148 mmol) followed by a solution of AIBN (2.2338 g solution
dissolved in DMF at a concentration of 1.0927 mg/g, (2.44 mg AIBN,
0.0148 mmol, 2,2'-azobis(2-methylpropionitrile), compound
recrystallized from MeOH) then an additional amount of DMF (2.6163
g) was added bringing the total amount of DMF used in this reaction
to 4.8501 g. This solution was mixed and vortexed for several
minutes until all of the CTA was completely dissolved. Once all the
CTA was completely dissolved then BMA (1.1849 g, 8.314 mmol,
purified by passing the neat monomer through a plug of
Al.sub.2O.sub.3, butyl methacrylate, d--0.894 g/mL), PAA (488.0 mg,
4.231 mmol, unpurified 2-propylacrylic acid, d--0.951 g/mL), DMAEMA
(661.8 mg, 4.231 mmol, purified by passing the neat monomer through
a plug of Al.sub.2O.sub.3, 2-(dimethylamino)ethyl methacrylate,
d--0.933 g/mL), and PDSMA (227.0 mg, 0891 mmol). This mixture was
mixed for several minutes. The reaction mixture was then
transferred to a brand new 20 mL reaction vial containing a
magnetic stir bar. The reaction vial was partially sealed and
cooled to 0.degree. C. using an ice bath while the mixture was
degassed by vigorously bubbling nitrogen for 30 minutes with
magnetic stirring of the reaction solution. The vial was then
completely sealed and placed into a heater block. The stirring
speed was set at 300, the thermometer was set at 62.degree. C. The
reaction was left to stir at 62.degree. C. for 5 hours and 50
minutes. After the reaction is complete it was quenched by opening
the vial and then placing the reaction vial in ice exposing the
mixture to air. The reaction solution was then diluted with acetone
(.about.5 mL, roughly the same volume of acetone as the DMF used in
the reaction vial) and precipitated into a stirred mixture of
Et.sub.2O/hexanes (1000 mL, 1:4 v/v) in a glass beaker. After the
polymer had settled to the bottom (ca. 15 min) the solvents were
decanted off. The precipitated polymer dissolved in MeOH was
transferred into dialysis membranes with a 2000 g/mol molecular
weight cut off (Spectrum Labs, Spectrum Spectra/Por 6 Dialysis
Membrane Tubing MWCO: 2000) and dialyzed against MeOH (3.times.4000
mL) for 3 days (72 h). The dialysis solution was changed every day
for 3 iterations total. After 3 days (72 h) dialysis against MeOH
the dialysis solution is changed to nanopure H.sub.2O and dialyzed
against H.sub.2O (5.times.4000 mL) for 5 hr. The dialysis solution
was changed roughly every hour for 5 iterations total. Upon
completion of dialysis the solutions were transferred to tared
vials and frozen solid using a bucket of dry ice. Then the material
was placed into the lyophilizer for >4 days total drying time.
This process provided 1.0325 g of the final product. The final
product was then analyzed by NMR and GPC. Analysis of the polymer
by .sup.1H-NMR indicated a polymer with no vinyl groups remaining
and the presence of PDSMA. The NMR is consistent for proposed
structure. GPC results: Mn=11.7 kDa, dn/dc=0.058046. The final
product was stored in glass vials with rubber septum that were
purged with argon and sealed with parafilm. The vials were stored
at -20.degree. C.
Example 17: Polymer Synthesis
[0561] By similar methods, the following polymers were synthesized
according to the following conditions shown in Tables 8-67, below.
[0562] A. P67: NAG-PEG12-[PEGMA(300,
79.1%)-BPAM(10.0%)-PDSMA(10.9%)]3.56
KDa-b-[DMAEMA(34.7%)-BMA(54.7%)-PAA(10.5%)]4.71 KDa
TABLE-US-00010 [0562] TABLE 8 P67 Block 1 Block 2 [M/CTA/I]
[12.8:1.6:1.6/1/0.05] [30:59:30/1/0.1] [concentration] 1.17M 2.61M
Time 1 h 45 m 5 h 35 m Temperature 67.degree. C. 61.degree. C. CTA
= Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0563] B. P68: NAG-PEG12-[PEGMA(300; 89.8%)-PhEMA(10.2%)]3.23
KDa-b-[DMAEMA(33%)-BMA(57%)-PAA(10%)]6.0 KDa
TABLE-US-00011 [0563] TABLE 9 P68 Block 1 Block 2 [M/CTA/I]
[13.95:1.55/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time
1 h 30 m 4 h 30 m Temperature 67.degree. C. 65.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0564] C. P69: NAG-PEG12-[PEGMA(300;78.7%)-PhEMA(21.3%)]3.25
KDa-b-[DMAEMA(32.9%)-BMA(54.8%)-PAA(12.3%)]5.4 KDa
TABLE-US-00012 [0564] TABLE 10 P69 Block 1 Block 2 [M/CTA/I]
[12.4:3.1/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1
h 30 m 4 h 30 m Temperature 67.degree. C. 65.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0565] D. P70: NAG-PEG12-[PEGMA(300,88.6)-PhEMA(11.4%)]3.02
KDa-b-[DMAEMA(36.8%)-BMA(56.3%)-PAA(6.9%)]4.39 KDa
TABLE-US-00013 [0565] TABLE 11 P70 Block 1 Block 2 [M/CTA/I]
[12.4:3.1/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1
h 30 m 4 h 30 m Temperature 67.degree. C. 65.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0566] E. P71: NAG-PEG12-[PEGMA(300, 69.5%)-BPAM (19.2%)-PDSMA
(11.3%)]3.59 KDa-b-[DMAEMA (35.2%)-BMA (53.9%)-PAA (10.9%)]5.27
Kda
TABLE-US-00014 [0566] TABLE 12 P71 Block 1 Block 2 [M/CTA/I]
[12.8:3.2:1.65/1/0.05] [30:59:30/1/0.1] [concentration] 1.22M 2.62M
Time 1 h 45 m 5 h 35 m Temperature 67.degree. C. 61.degree. C. CTA
= Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0567] F. P72: NAG-PEG12-[PEGMA(300, 80.3%)-ImMA(19.7)]3.7
KDa-b-[DMAEMA(35.9%)-BMA(53.9%)-PAA(10.2%)]4.7 KDa
TABLE-US-00015 [0567] TABLE 13 P72 Block 1 Block 2 [M/CTA/I]
[13:4.1/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1 h
30 m 5 h Temperature 67.degree. C. 65.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0568] G. P73: NAG-PEG12-[PEGMA(300,
73.1%)-BMA(14.4%)-PhEMA(12.5%)]3.8
KDa-b-[DMAEMA(37.6%)-BMA(52.3%)-PAA(10.1%)]4.2 KDa
TABLE-US-00016 [0568] TABLE 14 P73 Block 1 Block 2 [M/CTA/I]
[12.8:1.6:1.6/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M
Time 1 h 30 m 5 h Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0569] H. P74: NAG-PEG12-[PEGMA(300, 80.3%)-BMA(23.3%)]3.8
KDa-b-[DMAEMA(38.2%)-BMA(51.5%)-PAA(10.3%)]3.5 KDa
TABLE-US-00017 [0569] TABLE 15 P74 Block 1 Block 2 [M/CTA/I]
[12.8:3.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1
h 30 m 5 h Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0570] I. P75: NAG-PEG12-[PEGMA(300,
75.8%)-isoA-MA(11.8%)-PhEMA(12.4%)]3.3
KDa-b-[DMAEMA(39.3%)-BMA(51.6%)-PAA(9%)]4.95 KDa
TABLE-US-00018 [0570] TABLE 16 P75 Block 1 Block 2 [M/CTA/I]
[12.8:1.6:1.6/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M
Time 1 h 40 m 5 h Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0571] J. P76: NAG-PEG12-[PEGMA(300, 74.9%)-isoA-MA(25.1%)]2.9
KDa-b-[DMAEMA(38%)-BMA(53%)-PAA(9.1%)]5.2 KDa
TABLE-US-00019 [0571] TABLE 17 P76 Block 1 Block 2 [M/CTA/I]
[12.8:3.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1
h 40 m 5 h Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0572] K. P77: NAG-PEG12-[PEGMA (300, 86%)-CyHexMA (14%)]2.98
KDa-b-[DMAEMA (36.2%)-BMA (51.7%)-PAA (12.2%)]4.66 KDa
TABLE-US-00020 [0572] TABLE 18 P77 Block 1 Block 2 [M/CTA/I]
[12.8:2.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.6M Time
2 h 35 m 5 h Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0573] L. P78: NAG-PEG12-[PEGMA(300, 72.5%)-BPAM(27.5%)]3.8
KDa-b-[DMAEMA(25.6%)-BMA(64.8%)-PAA(9.6%)]5.5 KDa
TABLE-US-00021 [0573] TABLE 19 P78 Block 1 Block 2 [M/CTA/I]
[12.8:5/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1 h
45 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0574] M. P79: NAG-PEG12-[PEGMA (300, 69.9%)-HMA(30.1%)]2.93
KDa-b-[DMAEMA (34.4%)-BMA (536%)-PAA (12%)]4.43 Kda
TABLE-US-00022 [0574] TABLE 20 P79 Block 1 Block 2 [M/CTA/I]
[10.8:5.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.96M Time
1 h 50 m 4 h 40 m Temperature 68.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0575] N. P80: NAG-PEG12-[PEGMA (300, 85.4%)-EHMA(14.6%)]3.36
KDa-b-[DMAEMA (36.5%)-BMA (53.7%)-PAA (9.7%)]4.18 KDa
TABLE-US-00023 [0575] TABLE 21 P80 Block 1 Block 2 [M/CTA/I]
[16/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.62M Time 2 h 5
h Temperature 68.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0576] O. P81: NAG-PEG12-[PEGMA(300, 72%)-Fl-BMA(28%)]3.75
KDa-b-[DMAEMA(30.7%)-BMA(56.7%)-PAA(12.6%)]5.7 KDa
TABLE-US-00024 [0576] TABLE 22 P81 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0577] P. P82: NAG-PEG12-[PEGMA(300, 71.9%)-Fl-BMA(28.1%)]3.55
KDa-b-[DMAEMA(29.9%)-BMA(571.6%)-PAA(12.4%)]5.3 KDa
TABLE-US-00025 [0577] TABLE 23 P82 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0578] Q. P83: NAG-PEG12-[PEGMA(300, 78.9%)-F-CyHexMA(21.1%)]4.56
KDa-b-[DMAEMA(33.2%)-BMA(55.4%)-PAA(11.4%)]5.3 KDa
TABLE-US-00026 [0578] TABLE 24 P83 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0579] R. P84: NAG-PEG12-[PEGMA(300, 77.9%)-F-HPenMA(22.1%)]3.26
KDa-b-[DMAEMA(30.9%)-BMA(57.4%)-PAA(11.6%)]6.5 KDa
TABLE-US-00027 [0579] TABLE 25 P84 Block 1 Block 2 [M/CTA/I]
[12.8:4/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1 h
35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0580] S. P85: NAG-PEG12-[PEGMA(300, 79%)-BMA(21%)]2.9
KDa-b-[DMAEMA(29.3%)-BMA(26.6%)-Fl-BMA(34.6%)-PAA(9.5%)]5.8 KDa
TABLE-US-00028 [0580] TABLE 26 P85 Block 1 Block 2 [M/CTA/I]
[12.8:3.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1
h 40 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0581] T. P86: NAG-PEG12-[PEGMA (300, 78.1%)-C12MA(21.9%)]3.67
KDa-b-[DMAEMA (32.1%)-BMA (53.7%)-PAA (142%)]472 KDa
TABLE-US-00029 [0581] TABLE 27 P86 Block 1 Block 2 [M/CTA/I]
[12.8:3.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.38M Time
2 h 35 m 5 h 30 m Temperature 68.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0582] U. P87: NAG-PEG12-[PEGMA (300, 69.7%)-EHMA (30.3%)]3.9
KDa-b-[DMAEMA (31.1%)-BMA(56.7%)-PAA (12.1%)]51 KDa
TABLE-US-00030 [0582] TABLE 28 P87 Block 1 Block 2 [M/CTA/I]
[15.1:6.3/1/0.05] [30:59:30/1/0.1] [concentration] 1.24M 2.96M Time
2 h 15 m 6 h Temperature 68.degree. C. 62.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0583] V. P88: NAG-PEG12-[PEGMA (300, 76%)-5-NMA (24%)]3.0
KDa-b-[DMAEMA (34.4%)-BMA (54%)-PAA (11.6%)]5.6 KDa
TABLE-US-00031 [0583] TABLE 29 P88 Block 1 Block 2 [M/CTA/I]
[13.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.6M Time
2 h 6 h Temperature 67.degree. C. 61.5.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0584] W. P89: NAG-PEG12-[PEGMA (300,73.8%)-BMA (26.2%)]3.5
KDa-b-[DMAEMA (30.7%)-BMA(58.9%)-PAA (10.4%)]4.9 KDa
TABLE-US-00032 [0584] TABLE 30 P89 Block 1 Block 2 [M/CTA/I]
[13.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.29M 2.61M Time
2 h 5 m 5 h 45 m Temperature 69.degree. C. 61.degree. C. CTA =
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0585] X. P90: NAG-PEG12-[PEGMA (300, 72.6%)-HMA (27.4%)]3.58
KDa-b-[DMAEMA (30.6%)-BMA(56.2%)-PAA (13.3%)]5.6 KDa
TABLE-US-00033 [0585] TABLE 31 P90 Block 1 Block 2 [M/CTA/I]
[13:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.3M 2.72M Time 2
h 30 m 5 h 48 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0586] Y. P91: CH30-PEG12-[PEGMA (300, 92.8%)-PDSMA (7.2%)]3.6
KDa-b-[DMAEMA (34.2%)-BMA(54.7%)-PAA (11%)]6.5 KDa
TABLE-US-00034 [0586] TABLE 32 P91 Block 1 Block 2 [M/CTA/I]
[14:1.55/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.35M Time
1 h 45 m 5 h Temperature 67.degree. C. 65.5.degree. C. CTA
CH3O-PEG.sub.12-CTA; I = AIBN
[0587] Z. P92: NAG-PEG12-[PEGMA (300, 83.2%)-AEOMA (16.8%)]3.0
KDa-b-[DMAEMA (36.2%)-BMA(52.2%)-PAA (11.6%)]5.6 KDa
TABLE-US-00035 [0587] TABLE 33 P92 Block 1 Block 2 [M/CTA/I]
[12.8:2.2/1/0.05] [30:59:30/1/0.1] [concentration] 1.21M 2.5M Time
1 h 50 m 5 h 20 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0588] AA. P93: NAG-PEG12-[PEGMA (300, 77.6%)-CyHexMA (22.4%)]2.64
KDa-b-[DMAEMA (32.1%)-BMA(43.1%)-PAA (12.6%)-CyHexMA(12.3%)]4.67
KDa
TABLE-US-00036 [0588] TABLE 34 P93 Block 1 Block 2 [M/CTA/I]
[8.4:2.3/1/0.05] [30:45:30:10/1/0.1] [concentration] 1.21M 2.3M
Time 1 h 55 m 4 h Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0589] BB. P94: NAG-PEG12-[PEGMA (300, 72.2%)-B-Fl-HMA (27.8%)]4.2
KDa-b-[DMAEMA (35.7%)-BMA(54.4%)-PAA (9.9%)]4.7 KDa
TABLE-US-00037 [0589] TABLE 35 P94 Block 1 Block 2 [M/CTA/I]
[13:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1 h
35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0590] CC. P95: NAG-PEG12-[PEGMA(300, 71.2%)-Fl-BMA(28.8%)]3.55
KDa-b-[DMAEMA(34.2%)-BMA(57.9%)-PAA(7.9%)]4.9 KDa
TABLE-US-00038 [0590] TABLE 36 P95 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0591] DD. P96: NAG-PEG12-[PEGMA(300, 72.6%)-Fl-BMA(27.4%)]3.55
KDa-b-[DMAEMA(30.7%)-BMA(561%)PAA(13.2%)]4.9 KDa
TABLE-US-00039 [0591] TABLE 37 P96 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0592] EE. P97: NAG-PEG12-[PEGMA(300, 70.0%)-Fl-BMA(30.0%)]3.55
KDa-b-[DMAEMA(31.3%)-BMA(60.7%)-PAA(8.0%)]5.1 KDa
TABLE-US-00040 [0592] TABLE 38 P97 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0593] FF. P98: NAG-PEG12-[PEGMA(300, 75%)-2-Bu1-OMA(25%]4.26
KDa-b-[DMAEMA(32.1%)-BMA(55.7%)-PAA(12.2%)]5.69 KDa
TABLE-US-00041 [0593] TABLE 39 P98 Block 1 Block 2 [M/CTA/I]
[15:6.1/1/0.05] [30:59.5:30/1/0.1] [concentration] 1.3M 2.97M Time
2 h 30 m 5 h 45 m Temperature 70.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0594] GG. P99: NAG-PEG12-[PEGMA(300, 73.3%)-5-NMA(26.7%)]4.05
KDa-b-[DMAEMA(31.5%)-BMA(55.2%)-PAA(13.3%)]5.20 KDa
TABLE-US-00042 [0594] TABLE 40 P99 Block 1 Block 2 [M/CTA/I]
[15:6.1/1/0.05] [30:59:30/1/0.1] [concentration] 1.3M 2.76M Time 2
h 30 m 5 h 40 m Temperature 70.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0595] HH. P100: NAG-PEG12-[PEGMA(300, 74.1%)-Fl-BMA(25.9%)]3.79
KDa-b-[DMAEMA(29.9%)-BMA(56.2%)-PAA(13.9%)]5.44 KDa
TABLE-US-00043 [0595] TABLE 41 P100 Block 1 Block 2 [M/CTA/I]
[13:3.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.22M 2.52M Time 2
h 5 m 5 h 35 m Temperature 68.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0596] II. P101: NAG-PEG12-[PEGMA(300, 72.2%)-B-Fl-OMA(27.8%)]4.2
KDa-b-[DMAEMA(35.7%)-BMA(54.4%)-PAA(9.9%)]5.6 KDa
TABLE-US-00044 [0596] TABLE 42 P101 Block 1 Block 2 [M/CTA/I]
[13:5/1/0.05] [30:59:30/1/0.1] [concentration] 1.2M 2.3M Time 1 h
35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0597] JJ. P102: NAG-PEG12-[PEGMA(300, 71.9%)-F-BMA(28.1%)]3.55
KDa-b-[DMAEMA(27.3%)-BMA(60.9%)-PAA(11.9%)]4.55 KDa
TABLE-US-00045 [0597] TABLE 43 P102 Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 35 m 5 h 15 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0598] KK. P106: NAG-PEG12-[PEGMA(300, 74%)-HMA(26%)]4.1
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5 KDa
TABLE-US-00046 [0598] TABLE 44 P# Block 1 Block 2 [M/CTA/I]
[15.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.35M 2.3M Time
3 h 15 min 5 h 30 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0599] LL. P107: NAG-PEG12-[PEGMA(300, 74%)-HMA(26%)]*4.1
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*4.2 KDa
TABLE-US-00047 [0599] TABLE 45 P# Block 1 Block 2 [M/CTA/I]
[15.5:4.5/1/0.05] [27:51:36.5/1/0.1] [concentration] 1.35M 2.3M
Time 3 h 15 m 5 h 30 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0600] MM. P108: NAG-PEG12-[PEGMA(300, 80%)-HMA(20%)]*4.96
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5.5 KDa
TABLE-US-00048 [0600] TABLE 46 P# Block 1 Block 2 [M/CTA/I]
[19.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.5M 2.3M Time 3
h 10 m 6 h 10 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0601] NN. P109: NAG-PEG12-[PEGMA(300, 80%)-HMA(20%)]*4.96
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*6.5 KDa
TABLE-US-00049 [0601] TABLE 47 P# Block 1 Block 2 [M/CTA/I]
[19.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.5M 2.9M Time 3
h 10 m 7 h Temperature 69.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0602] OO. P110: NAG-PEG12-[PEGMA(300, 77.7%)-EHMA(22.3%)]4.37
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*6 KDa
TABLE-US-00050 [0602] TABLE 48 P# Block 1 Block 2 [M/CTA/I]
[16:5/1/0.05] [30:59:30/1/0.1] [concentration] 1.3M 3M Time 3 h 6 h
30 m Temperature 68.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0603] PP. Pill: NAG-PEG12-[PEGMA(300, 77%)-Fl-BMA(23%)]5.80
KDa-b-[DMAEMA(27.3%)-BMA(60.9%)-PAA(11-9%)]*5.74 KDa
TABLE-US-00051 [0603] TABLE 49 P# Block 1 Block 2 [M/CTA/I]
[20:4.3/1/0.05] [30:59:30/1/0.1] [concentration] 1.5M 2.3M Time 3 h
6 h 20 m Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0604] QQ. P112: NAG-PEG12-[PEGMA(300, 77%)-Fl-BMA(23%)]5.80
KDa-b-[DMAEMA(27.3%)-BMA(60.9%)-PAA(11.9%)]*6.10 KDa
TABLE-US-00052 [0604] TABLE 50 P# Block 1 Block 2 [M/CTA/I]
[20:4.3/1/0.05] [30:59:30/1/0.1] [concentration] 1.5M 2.3M Time 3 h
7 h 20 m Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0605] RR. P113: NAG-PEG12-[PEGMA(300, 84.9%)-Chol-MA(15.1%)]*3.5
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*4.67 KDa
TABLE-US-00053 [0605] TABLE 51 P# Block 1 Block 2 [M/CTA/I]
[12.8:2.2/1/0.05] [26:51:26/1/0.1] [concentration] 0.97M 2.3M Time
2 h 15 m 5 h Temperature 67.degree. C. 63.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0606] SS. P114: NAG-PEG12-[PEGMA(300, 67%)-HMA(33%)]*5.7
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*6.15 KDa
TABLE-US-00054 [0606] TABLE 52 P# Block 1 Block 2 [M/CTA/I]
[20:7.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.55M 2.89M Time 4
h 5 h 45 m Temperature 68.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0607] TT. P115: NAG-PEG12-[PEGMA(300, 67%)-HMA(33%)]*5.7
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*6 KDa
TABLE-US-00055 [0607] TABLE 53 P# Block 1 Block 2 [M/CTA/I]
[20:7.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.55M 2.89M Time 4
h 7 h Temperature 68.degree. C. 62.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0608] UU. P116: NAG-PEG12-[PEGMA(300, 73%)-F1-BMA(27%)]*+6.3
KDa-b-[DMAEMA(27.3%)-BMA(60.9%)-PAA(11.9%)]*+5.9 KDa
TABLE-US-00056 [0608] TABLE 54 P# Block 1 Block 2 [M/CTA/I]
[20:6.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.5M 2.3M Time 3 h
7 h Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
+Molecular weight of block is estimated based on trace overlays
with polymers of known molecular weight
[0609] VV. P117:
[0609] ##STR00036## [0610] NAG-PEG 12-[PEGMA(300,
72%)-PF-BMA(28%)]*+3.7
KDa-b-[DMAEMA(27.3%)-BMA(60.9%)-PAA(11.9%)]*5.0 KDa
TABLE-US-00057 [0610] TABLE 55 P# Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.5M 2.3M Time 1
h 45 min 5 h 20 min Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
+Molecular weight of block is estimated based on trace overlays
with polymers of known molecular weight
[0611] WW. P118: NAG-PEG12-[PEGMA(300, 70%)-HMA(30%)]*5.2
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5.7 KDa
TABLE-US-00058 [0611] TABLE 56 P# Block 1 Block 2 [M/CTA/I]
[20:7/1/0.05] [30.7:60:30.7/1/0.1] [concentration] 1.5M 2.3M Time 3
h 15 m 5 h 45 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0612] XX. P119: NAG-PEG12-[PEGMA(300, 70%)-HMA(30%)]*5.2
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5 KDa
TABLE-US-00059 [0612] TABLE 57 P# Block 1 Block 2 [M/CTA/I]
[20:7/1/0.05] [26:52:26/1/0.1] [concentration] 1.5M 2.3M Time 3 h
15 m 5 h 25 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0613] YY. P120: NAG-PEG12-[PEGMA(300, 75%)-CyHexMA(25%)]*4
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5.2 KDa
TABLE-US-00060 [0613] TABLE 58 P# Block 1 Block 2 [M/CTA/I]
[15.5:4.5/1/0.05] [30.7:60:30.7/1/0.1] [concentration] 1.3M 2.3M
Time 3 h 5 h 40 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0614] ZZ. P121: NAG-PEG12-[PEGMA(300, 75%)-Me-CyHexMA(25%)]*4.3
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(139c)]*5.1 KDa
TABLE-US-00061 [0614] TABLE 59 P# Block 1 Block 2 [M/CTA/I]
[16:4/1/0.05] [30.7:60:30.7/1/0.1] [concentration] 1.3M 2.3M Time 3
h 5 h 35 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0615] AAA P122: NAG-PEG12-[PEGMA(300, 73%)-Fl-BMA(27%)]*+6.3
KDa-b-[DMAEMA(27.3%)-BMA(60.9%)-PAA(11.9%)]*+6.9 KDa
TABLE-US-00062 [0615] TABLE 60 P# Block 1 Block 2 [M/CTA/I]
[20:6.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.5M 2.6M Time 3 h
9 h Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
+Molecular weight of block is estimated based on trace overlays
with polymers of known molecular weight
[0616] BBB. P123: NAG-PEG12-[PEGMA(300, 79%)-Bu1-O-MA(21%)]*4.88
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*4.6 KDa
TABLE-US-00063 [0616] TABLE 61 P# Block 1 Block 2 [M/CTA/I]
[16:4/1/0.05] [30.7:60:30.7/1/0.1] [concentration] 1.3M 2.3M Time 3
h 30 m 5 h 20 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0617] CCC. P124: NAG-PEG12-[PEGMA(300, 74%)-HMA(26%)]*4.15
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5 KDa
TABLE-US-00064 [0617] TABLE 62 P# Block 1 Block 2 [M/CTA/I]
[15.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.35M 2.3M Time
3 h 15 min 5 h 30 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0618] DDD P125: NAG-PEG12-[PEGMA(300, 74%)-HMA(26%)]*4.15
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5 KDa
TABLE-US-00065 [0618] TABLE 63 P# Block 1 Block 2 [M/CTA/I]
[15.5:4.5/1/0.05] [30:59:30/1/0.1] [concentration] 1.35M 2.3M Time
3 h 15 min 5 h 30 m Temperature 69.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0619] EEE. P103: NAG-PEG12-[PEGMA(300, 70.3%)-Fl-BMA(29.7%)]3.6
KDa-b-[DMAEMA(32.2%)-BMA(57.6%)-PAA(10.2%)]5 KDa
TABLE-US-00066 [0619] TABLE 64 P# Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:52:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 42 m 5 h 30 m Temperature 68.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN
[0620] FFF. P104: NAG-PEG12-[PEGMA(300, 68%)-Fl-BMA(32%)]*3.7
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*5.3 KDa
TABLE-US-00067 [0620] TABLE 65 P# Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 40 min 5 h 30 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
[0621] GGG P105: NAG-PEG12-[PEGMA(300, 73%)-F1-BMA(27%)]*+4.3
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*+5.3 KDa
TABLE-US-00068 [0621] TABLE 66 P# Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 40 min 5 h 30 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
+Molecular weight of block is estimated based on trace overlays
with polymers of known molecular weight
[0622] HHH P106: NAG-PEG12-[PEGMA(300, 73%)-F1-BMA(27%)]*+4.3
KDa-b-[DMAEMA(31%)-BMA(56%)-PAA(13%)]*+5.3 KDa
TABLE-US-00069 [0622] TABLE 67 P# Block 1 Block 2 [M/CTA/I]
[12.8:3.5/1/0.05] [26:51:26/1/0.1] [concentration] 1.2M 2.3M Time 1
h 40 min 5 h 30 m Temperature 67.degree. C. 61.degree. C. CTA
Nag(OH)C5N-PEG.sub.12-CTA; I = AIBN *Monomer incorporation for
block is estimated based on historical incorporation levels
+Molecular weight of block is estimated based on trace overlays
with polymers of known molecular weight
Example 18: In Vivo Expression of mRNA with Lipid-mRNA Formulations
and Co-Injection or Sequential Injection of Additional Polymers
[0623] Additional polymers were tested with sequential or
co-injection with mRNA/LNP using the same methods as described in
Example 2.
[0624] Table 68 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticles with sequential injection of polymer P1435, P1299, or
P67 at 1 minute following the first injection. Data was acquired at
6 hours post dose. mRNA/LNP+polymer P67 showed 5-fold and 8-fold
improvement in luminescent signal compared to polymers P1435 or
P1299, respectively.
TABLE-US-00070 TABLE 68 Timing mRNA Total Flux Lipid-mRNA Fluc
Between Dose (photons/sec) Nanoparticle Polymer mRNA Injections
(mg/kg) Geomean STDEV Buffer None None NA 0 1.83E+05 NA
DOTAP:CHEMS: P1435 Fluc 3 1 min 1 1.23E+09 1.15E+09 CHOL:DMPE- 75
mg/kg mRNA PEG2K P1299 Fluc 3 1 7.80E+08 2.19E+09 (50:32:16:2) 75
mg/kg mRNA N:P 7 26 mg/kg P67 Fluc 3 1 6.23E+09 7.28E+09 75 mg/kg
mRNA
[0625] Table 69 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticles with sequential injection of polymer P67 at 1 minute
following the first injection. Data was acquired at 6 hours post
dose. In this study, two different Fluc mRNAs were tested. Fluc 2
mRNA showed a 21-fold improvement in luminescence signal compared
to Fluc 1 mRNA. Modifications of Fluc 1 and Fluc 2 mRNAs are
described above in Example 2.
TABLE-US-00071 TABLE 69 Timing mRNA Total Flux Lipid-mRNA Between
Dose (photons/sec) Nanoparticle Polymer Fluc mRNA Injections
(mg/kg) Geomean STDEV Buffer None None NA 0 2.81E+05 NA
DOTAP:CHEMS: P67 Fluc 1 1 min 1 4.20E+08 1.82E+08 CHOL:DMPE- 75
mg/kg mRNA PEG2K Fluc 2 1 8.85E+09 3.90E+09 (50:32:16:2) mRNA N:P 7
26 mg/kg
[0626] Table 70 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticles with co-injection of NAG targeted polymer P67
compared to non-targeted polymer P91. mRNA/LNP+polymer were mixed
at a 1:1 ratio and injected immediately into mice. Data was
acquired at 6 hours post dose. mRNA/LNP+NAG targeted polymer P67
showed 130-fold improvement in luminescent signal compared to
non-targeted polymer P91.
TABLE-US-00072 TABLE 70 Timing mRNA Total Flux Lipid-mRNA Fluc
Between Dose (photons/sec) Nanoparticle Polymer mRNA Injections
(mg/kg) Geomean STDEV Buffer None None NA 0 2.03E+05 DOTAP:CHEMS:
P67 Fluc 2 co- 1 4.03E+09 7.04E+09 CHOL:DMPE- 75 mg/kg mRNA
injection PEG2K P91 Fluc 2 1 3.07E+07 9.45E+06 (50:32:16:2) 75
mg/kg mRNA N:P 7 26 mg/kg
Example 19: DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k and
DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k-NAG mRNA Nanoparticle
Formulation with Sequential or Co-Injection of a Polymer:
Formulation Characteristics
[0627] DOTAP (Corden Pharma, Boulder, Colo., USA; catalog number
LP-R4-117) was solubilized at 50 mg/mL in 200 proof ethanol at room
temperature for 15 minutes. The DSPE-PEG.sub.2K(Corden Pharma,
Boulder, Colo., USA; catalog number LP-R4-039) or the DSPE-PEG-NAG
(PhaseRx Inc.) was solubilized at 50 mg/mL in 200 proof ethanol at
room temperature for 15 minutes. The cholesteryl hemisuccinate
(CHEMS) (Avanti Polar Lipid Alabaster, Ala., USA; catalog number
850524P) and the Cholesterol (CHOL) (Corden Pharma, Boulder, Colo.,
USA; catalog number CH-0355) were individually solubilized at 25
mg/mL in 200 proof at 75.degree. C. for 5 minutes. Typically, for a
2 mL preparation of DOTAP:CHEMS:CHOL:DSPE-PEG.sub.2K (50:32:8:10
mol %) LNP at a N:P ratio of 7, a lipid ethanolic mixture
containing 178 .mu.L of DOTAP at 50 mg/mL in 200 proof ethanol, 158
.mu.L of CHEMS at 25 mg/mL in 200 proof ethanol, 31 .mu.L of CHOL
at 25 mg/mL in 200 proof ethanol, 143 .mu.L of DSPE-PEG.sub.2K at
50 mg/mL in 200 proof ethanol and 156 .mu.L of 200 proof ethanol
was prepared for a final volume of 0.666 mL and a total lipid
concentration of 31 mg/mL. For 2 mL preparation of
DOTAP:CHEMS:CHOL:DSPE-PEG.sub.2K-NAG (50:32:8:10 mol %) LNP at a
N:P ratio of 7, the lipid ethanolic mixture containing 178 .mu.L of
DOTAP at 50 mg/mL in 200 proof ethanol, 158 .mu.L of CHEMS at 25
mg/mL in 200 proof ethanol, 31 .mu.L of CHOL at 25 mg/mL in 200
proof ethanol, 160 .mu.L of DSPE-PEG.sub.2K-NAG at 50 mg/mL in 200
proof ethanol and 161 .mu.L of 200 proof ethanol was prepared for a
final volume of 0.666 mL and a total lipid concentration of 32.5
mg/mL.
[0628] The lipid nanoparticle (LNP) formulations were prepared at
N:P (nitrogen to phosphate) ratios from 1.75 to 14 based on the
DOTAP concentration. The DOTAP:CHEMS ratio was fixed at 1.6 at
50:32 mol % respectively at the various N:P ratios. DSPE-PEG.sub.2K
or DSPE-PEG.sub.2K-NAG were varied from 1 to 15 mol %. The CHOL mol
% was adjusted to result in 100 mol % final lipid
concentration.
[0629] The Fluc (firefly luciferase) mRNA stock solution at 1 mg/mL
in 10 mM Tris-HCl (pH 7.5) was diluted to 0.45 mg/mL in 300 mM
sucrose 20 mM phosphate, pH 7.4 buffer (SUP buffer). The mRNA/LNPs
were assembled at N:P ratios from 1.75 to 14 by mixing the
ethanolic lipid solution with 0.45 mg/mL mRNA in SUP buffer at a
1:2 ratio (lipid ethanolic mixture:mRNA in SUP buffer) using the
microfluidic device from Precision NanoSystems Inc (Vancouver BC,
Canada) at a 12 mL/minute flow rate. The mRNA/LNPs in 33% ethanol
were then incubated at room temperature for 60 minutes prior to
dialysis for 18 hours against 100 volumes (200 mL) of SUP
buffer.
[0630] The polymers used for sequential injection or co-injection
were solubilized at 20 mg/mL in SUP buffer with agitation at 400
rpm for 1 hour and then stored overnight at 4.degree. C. The
polymers were diluted to 5-10 mg/mL in SUP buffer prior to
injection.
[0631] If mRNA/LNP and polymer were co-injected, a 2.times.
solution of each was prepared. Just prior to dosing, the solutions
were mixed and injected immediately.
[0632] The formulation particle size was measured by adding 10
.mu.L of formulation to 90 .mu.L of SUP buffer into a disposable
micro-cuvette and analyzed using the Malvern Instrument ZETASIZER
NANO-ZS. The LNPs showed a particle size of 85 nm (Z-average). The
formulation zeta-potential at pH 7.4 was measured by adding 10
.mu.L of formulation to 740 .mu.L of SUP buffer into a disposable 1
mL cuvette. The formulation zeta-potential at pH 4 was measured by
adding 10 .mu.L of formulation to 740 .mu.L of sucrose acetate
buffer (pH 4) into a disposable 1 mL cuvette. The zeta dip cell was
inserted into the 1 mL cuvette and the formulation was analyzed
using the ZETASIZER NANO-ZS. Typically, the DOTAP LNPs had a zeta
potential of +1.6 mV at pH 7 and +10 mV at pH 4.0. The ability of
the LNP to compact the mRNA was measured in a 96 well plate using a
SYBR Gold dye accessibility assay. Typically, 50 .mu.L of the lipid
formulation at 0.01 mg/mL mRNA was added to 150 .mu.L of diluted
SYBR Gold stock solution (1 .mu.L of Stock SYBR Gold in 3 mL of SUP
buffer) and incubated for 15 minutes at room temperature with
agitation (100 RPM). The fluorescence was read at an excitation
wavelength of 495 nm and emission wavelength of 538 nm. The percent
dye accessibility was calculated by dividing the fluorescence
intensity of the formulated mRNA by the fluorescence intensity of
the free mRNA.times.100. The DOTAP LNPs showed 8% dye accessibility
when prepared in SUP buffer. Table 71 below shows characterization
of exemplary LNP formulations.
TABLE-US-00073 TABLE 71 LNPs Characteristics Sample # RP600-1
RP495-13 Lipid DOTAP:CHEMS: DOTAP:CHEMS: CHOL:DSPE- CHOL:DSPE-
PEG2K PEG2K-NAG (50:32:8:10) (50:32:8:10) N/P 7 7 Lipid
Concentration (mg/mL) 9.5 10.8 Visual Appearance Opalescent (+)
Opalescent (+) % Dye access SUP pH 7.4 8% 8% Z-Ave (nm) 85 98 PDI
0.242 0.312 Number (nm) 38 37 Pk 1 Mean Int (nm) 105 232 Pk 2 Mean
Int (nm) 4536 63 Pk 1 Area Int (%) 97 57 Pk 2 Area Int (%) 3 43 ZP
pH 7.4 (mV) 1.6 -5 ZP pH 4 (mV) 10 8 Sizing data quality Good
Good
Example 20: In Vivo Expression of mRNA with
DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k and
DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k-NAG mRNA Formulations and
Co-Injection or Sequential Injection of Polymer
[0633] Additional LNPs described in Example 19 were tested with
various polymers using sequential or co-injection and the same
methods as described in Example 2.
[0634] Table 72 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k,
DOTAP:CHEMS:CHOL:DSPE-PEG2k, or
DOTAP:CHEMS:CHOL:DSPE-PEG2k-NAG+Fluc mRNA nanoparticles with
co-injection of polymer P67. mRNA/LNP+polymer were mixed at a 1:1
ratio and injected immediately into mice. Data was acquired at 6,
24, and 48 hours post dose. Both DOTAP:CHEMS:CHOL:DSPE-PEG2k-NAG
and DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP showed longer duration of
expression with 8.7-fold and 2.6-fold greater luminescent signal in
area under the curve (AUC) values compared to
DOTAP:CHEMS:CHOL:DMPE-PEG2k LNP respectively.
TABLE-US-00074 TABLE 72 Fluc 2 Fold mRNA Imaging Total Flux Change
to Lipid-mRNA Dose Time (photons/sec) DMPE- Nanoparticle Polymer
(mg/kg) Point Geomean STDEV AUC PEG2K LNP Buffer None 0 6 h
3.34E+05 DOTAP:CHEMS: P67 1 6 h 3.61E+09 7.87E+08 4.02E+10 1.0
CHOL:DMPE- 75 mg/kg 24 h 3.17E+08 1.23E+08 PEG2K 48 h 1.11E+07
3.07E+06 (50:32:16:2) N:P 7 26 mg/kg DOTAP:CHEMS: P67 1 6 h
7.23E+09 3.87E+09 1.05E+11 2.6 CHOL:DSPE- 75 mg/kg 24 h 1.16E+09
1.08E+09 PEG2K 48 h 2.15E+08 9.83E+07 (50:32:8:10) N:P 7 35 mg/kg
DOTAP:CHEMS: P67 1 6 h 1.51E+10 2.15E+10 3.49E+11 8.7 CHOL:DSPE- 75
mg/kg 24 h 4.14E+09 6.19E+09 PEG2K-NAG 48 h 1.19E+08 2.03E+08
(50:32:8:10) N:P 7 36 mg/kg
[0635] Table 73 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k or
DOTAP:CHEMS:CHOL:DSPE-PEG2k-NAG+Fluc mRNA nanoparticles with
co-injection of polymer P71 or P81. mRNA/LNP+polymer were mixed at
a 1:1 ratio and injected immediately into mice. Data was acquired
at 6, 24, 48, 72, and 96 hours post dose.
DOTAP:CHEMS:CHOL:DSPE-PEG2k and DOTAP:CHEMS:CHOL:DSPE-PEG2k-NAG
LNPs+P81 showed 7-fold and 2.8-fold greater luminescent signal in
area under the curve (AUC) values compared to either LNP+P71
respectively.
TABLE-US-00075 TABLE 73 Fluc 2 mRNA Imaging Total Flux Lipid-mRNA
Dose Time (photons/sec) Nanoparticle Polymer (mg/kg) Point Geomean
STDEV AUC Buffer None 0 6 h 1.35E+05 DOTAP:CHEMS:CHOL: P71 1 6 h
1.19E+10 9.52E+09 2.88E+11 DSPE-PEG2K 50 mg/kg 24 h 5.45E+09
3.96E+09 (50:32:8:10) 48 h 9.81E+07 8.21E+07 N:P 7 35 mg/kg 72 h
6.66E+06 4.99E+06 96 h 1.86E+06 1.17E+06 DOTAP:CHEMS:CHOL: P81 1 6
h 9.40E+10 5.40E+10 2.01E+12 DSPE-PEG2K 45 mg/kg 24 h 3.26E+10
3.23E+10 (50:32:8:10) 48 h 6.91E+08 7.29E+08 N:P 7 35 mg/kg 72 h
4.28E+07 4.38E+07 96 h 1.05E+07 9.19E+06 DOTAP:CHEMS:CHOL: P71 0.5
6 h 2.17E+10 1.88E+10 3.95E+11 DSPE-PEG2K- 50 mg/kg 24 h 5.84E+09
4.45E+09 NAG (50:32:8:10) 48 h 8.88E+07 9.94E+07 N:P 7 36 mg/kg 72
h 7.06E+06 6.69E+06 96 h 2.10E+06 2.09E+06 DOTAP:CHEMS:CHOL: P81
0.5 6 h 6.06E+10 1.16E+10 7.95E+11 DSPE-PEG2K- 35 mg/kg 24 h
9.87E+09 6.23E+09 NAG (50:32:8:10) 48 h 1.60E+08 1.33E+08 N:P 7 36
mg/kg 72 h 1.21E+07 7.43E+06 96 h 3.91E+06 2.24E+06
[0636] Table 74 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc 2 mRNA
nanoparticles with co-injection of polymer P71 or P92.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P71 showed 4 to 13-fold
greater luminescent signal compared to P92.
TABLE-US-00076 TABLE 74 Fluc Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 1 P71 50 5.97E+09 8.09E+09
DSPE-PEG2K 1 P92 25 4.71E+08 7.35E+08 (50:32:8:10) 1 P92 50
1.37E+09 1.62E+09 N:P 7 35 mg/kg
[0637] Table 75 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc 2 mRNA
nanoparticles with co-injection of polymer P71, P93, P79, or P80.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P80 or P79 showed 5-fold or
2-fold greater luminescent signal compared to P71 respectively. P93
showed similar activity to P71.
TABLE-US-00077 TABLE 75 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.1 P71 50 4.74E+08 3.69E+08
DSPE-PEG2K 0.1 P93 25 2.04E+08 2.05E+08 (50:32:8:10) 0.1 P93 50
3.41E+08 3.65E+08 N:P 7 3.5 mg/kg 0.1 P79 25 1.12E+09 4.36E+08 0.1
P80 25 2.37E+09 1.93E+09
[0638] Table 76 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc 2 mRNA
nanoparticles with co-injection of polymer P71, P82, P94, or P86.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P82, P94, or P86 showed 6 to
13-fold greater luminescent signal compared to P71.
TABLE-US-00078 TABLE 76 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P71 50 1.61E+09 1.75E+09
DSPE-PEG2K 1 P82 30 1.62E+10 6.45E+09 (50:32:8:10) 1 P82 40
1.53E+10 1.80E+10 N:P 7 35 mg/kg 1 P94 40 2.01E+10 7.91E+09 1 P86
40 1.00E+10 1.21E+10
[0639] Table 77 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc 2 mRNA
nanoparticles with co-injection of polymer P71, P87, P88, or P89.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P87, P88, or P89 showed 3 to
18-fold greater luminescent signal compared to P71.
TABLE-US-00079 TABLE 77 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.1 P71 50 1.54E+08 1.23E+08
DSPE-PEG2K 0.1 P87 25 4.05E+08 7.71E+08 (50:32:8:10) 0.1 P87 35
2.85E+09 3.22E+09 N:P 7 3.5 mg/kg 0.1 P88 25 1.26E+09 1.87E+09 0.1
P89 25 3.89E+08 2.19E+08 0.1 P89 35 6.06E+08 6.54E+08 0.1 P89 50
1.11E+09 9.00E+08
[0640] Table 78 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc 2 mRNA
nanoparticles with co-injection of polymer P95, P90, P96, or P87.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Fluc
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P90, P96, or P87 showed
similar luminescent signal as P95.
TABLE-US-00080 TABLE 78 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 1 P95 30 1.17E+10 1.34E+10
DSPE-PEG2K 1 P95 40 4.18E+10 2.54E+10 (50:32:8:10) 1 P96 35
2.09E+10 2.35E+10 N:P 7 35 mg/kg 1 P90 30 1.59E+10 1.78E+10 1 P87
35 3.27E+10 1.39E+10
[0641] Table 79 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P71, P77, or P78.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Fluc
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P77 or P78 showed 3 to 8-fold
greater luminescent signal compared to P71.
TABLE-US-00081 TABLE 79 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P71 50 1.10E+09 1.02E+09
DSPE-PEG2K 0.5 P77 25 1.90E+09 1.01E+09 (50:32:8:10) 0.5 P77 50
1.12E+09 2.37E+09 N:P 7 17 mg/kg 0.5 P77 75 9.02E+09 1.00E+10 0.5
P78 25 3.46E+08 3.56E+08 0.5 P78 50 3.78E+09 1.85E+09
[0642] Table 80 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P96, P98, P99, or P100.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P98, P99, or P100 showed 3 to
5-fold greater luminescent signal compared to P96.
TABLE-US-00082 TABLE 80 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.1 P96 35 1.30E+09 1.17E+09
DSPE-PEG2K 0.1 P98 25 1.23E+09 2.46E+09 (50:32:8:10) 0.1 P98 35
4.62E+09 2.14E+09 N:P 7 3.5 mg/kg 0.1 P99 25 5.80E+09 1.54E+09 0.1
P100 25 1.22E+09 2.18E+09 0.1 P100 35 3.24E+09 5.98E+09
[0643] Table 81 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P82, P90, P106, or P107.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P90, P106, or P107 showed 3 to
10-fold greater luminescent signal compared to P82.
TABLE-US-00083 TABLE 81 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P82 30 3.78E+09 9.23E+09
DSPE-PEG2K 0.5 P90 25 7.12E+09 3.69E+09 (50:32:8:10) 0.5 P90 35
2.74E+10 8.39E+09 N:P 7 17.5 mg/kg 0.5 P106 25 1.85E+10 1.43E+10
0.5 P106 35 4.12E+10 1.26E+10 0.5 P106 45 1.65E+10 3.47E+10 0.5
P107 25 7.93E+09 4.97E+09 0.5 P107 35 1.47E+10 9.46E+09 0.5 P107 45
1.35E+10 1.34E+10
[0644] Table 82 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P97, P104, P108, or
P109. mRNA/LNP+polymer were mixed at a 1:1 ratio and injected
immediately into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P104, P108, or P109 showed up
to 2-fold greater luminescent signal compared to P97.
TABLE-US-00084 TABLE 82 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P97 30 1.08E+10 5.89E+09
DSPE-PEG2K 0.5 P104 25 4.49E+09 9.32E+08 (50:32:8:10) 0.5 P104 30
6.82E+09 2.69E+10 N:P 7 17.5 mg/kg 0.5 P104 35 2.58E+10 3.59E+09
0.5 P108 25 1.37E+10 1.40E+10 0.5 P108 35 1.36E+10 1.58E+10 0.5
P108 45 2.37E+10 2.28E+10 0.5 P109 25 8.33E+09 1.25E+10 0.5 P109 35
2.07E+10 2.31E+10
[0645] Table 83 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P103, P90, P106, or
P108. mRNA/LNP+polymer were mixed at a 1:1 ratio and injected
immediately into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P90, P106, or P108 showed up
to 2-fold greater luminescent signal compared to P103.
TABLE-US-00085 TABLE 83 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P103 30 5.94E+10 3.36E+10
DSPE-PEG2K 0.5 P103 35 7.11E+10 4.71E+10 (50:32:8:10) 0.5 P90 30
1.52E+10 2.78E+10 N:P 7 17.5 mg/kg 0.5 P90 35 7.65E+09 2.03E+10 0.5
P106 30 1.18E+11 2.23E+10 0.5 P106 35 4.94E+10 4.68E+10 0.5 P108 30
9.45E+10 2.12E+10 0.5 P108 35 4.99E+10 5.03E+10
[0646] Table 84 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P95, P111, or P112.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Fluc
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P111 or P112 showed up to
4-fold greater luminescent signal compared to P95.
TABLE-US-00086 TABLE 84 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P95 30 6.19E+09 1.71E+10
DSPE-PEG2K 0.5 P111 25 4.12E+09 6.41E+09 (50:32:8:10) 0.5 P111 35
1.90E+10 3.63E+09 N:P 7 17.5 mg/kg 0.5 P112 25 7.28E+09 1.15E+10
0.5 P112 35 1.98E+10 1.49E+10 0.5 P112 45 2.66E+10 1.46E+10
[0647] Table 85 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P103, P106, P114 or
P115. mRNA/LNP+polymer were mixed at a 1:1 ratio and injected
immediately into mice. Data was acquired at 6 hours post dose. Fluc
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P106, P114, or P115 showed up
to 7-fold greater luminescent signal compared to P103.
TABLE-US-00087 TABLE 85 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P103 30 3.54E+09 5.27E+09
DSPE-PEG2K 0.5 P106 20 6.96E+09 4.36E+09 (50:32:8:10) 0.5 P106 25
1.19E+10 1.10E+10 N:P 7 17.5 mg/kg 0.5 P114 25 2.46E+10 1.16E+10
0.5 P115 25 8.28E+09 1.93E+10
[0648] Table 86 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P103, P116 or P117.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P116 or P117 showed lower
luminescent signal compared to P103.
TABLE-US-00088 TABLE 86 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P103 30 2.72E+10 1.13E+10
DSPE-PEG2K 0.5 P116 25 5.31E+09 3.32E+09 (50:32:8:10) 0.5 P116 35
1.20E+10 9.23E+09 N:P 7 17.5 mg/kg 0.5 P117 25 5.53E+08 5.10E+08
0.5 P117 35 1.35E+09 1.44E+09
[0649] Table 87 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P105, P98 or P123.
mRNA/LNP+polymer were mixed at a 1:1 ratio and injected immediately
into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P98 or P123 showed similar
luminescent signal compared to P105.
TABLE-US-00089 TABLE 87 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P105 30 2.11E+10 2.54E+10
DSPE-PEG2K 0.5 P98 20 1.85E+10 1.60E+10 (50:32:8:10) 0.5 P98 30
7.79E+09 1.93E+10 N:P 7 17.5 mg/kg 0.5 P98 40 2.07E+10 3.92E+10 0.5
P123 20 3.21E+10 1.56E+10 0.5 P123 30 2.77E+10 3.78E+10 0.5 P123 40
3.50E+10 3.16E+10
[0650] Table 88 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P105, P106, P124 or
P125. mRNA/LNP+polymer were mixed at a 1:1 ratio and injected
immediately into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P106, P124 or P125 showed up
to 2-fold greater luminescent signal compared to P105.
TABLE-US-00090 TABLE 88 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P105 30 1.80E+10 1.00E+10
DSPE-PEG2K 0.5 P106 25 6.46E+09 1.85E+10 (50:32:8:10) 0.5 P124 15
1.34E+10 2.10E+09 N:P 7 17.5 mg/kg 0.5 P124 25 4.16E+10 2.27E+10
0.5 P125 15 6.31E+09 9.98E+09 0.5 P125 25 3.79E+10 2.02E+10
[0651] Table 89 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+FLuc 2 mRNA
nanoparticles with co-injection of polymer P105, P118, P119 or
P110. mRNA/LNP+polymer were mixed at a 1:1 ratio and injected
immediately into mice. Data was acquired at 6 hours post dose. Flue
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P118, P119 or P110 showed
similar luminescent signal compared to P105.
TABLE-US-00091 TABLE 89 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P105 30 2.78E+10 1.32E+10
DSPE-PEG2K 0.5 P118 20 1.99E+10 6.98E+09 (50:32:8:10) 0.5 P118 30
2.86E+10 1.66E+10 N:P 7 17.5 mg/kg 0.5 P119 20 2.36E+10 8.30E+09
0.5 P119 30 2.42E+10 1.07E+10 0.5 P110 20 9.48E+09 1.10E+10 0.5
P110 30 2.22E+10 1.95E+10
Example 21: Therapeutic Efficacy of mRNA with Lipid-mRNA
Formulations and Co-Injection of Polymer in Ornithine
Transcarbamylase Deficient Mice
[0652] Hyperammonemia was induced in OTC-spf.sup.ash mice that were
treated with AAV2/8 vector/OTC shRNA to knockdown residual
endogenous OTC expression and activity (Cunningham et al., Mol Ther
19: 854-859, 2011). Plasma ammonia levels and orotic acid levels
were elevated in these mice. Four (4) days after AAV dosing, 1
mg/kg of OTC mRNA formulated in DOTAP:CHEMS:CHOL:DMPE-PEG.sub.2k
(50:32:16:2) at N:P 7+co-injection of 50 mg/kg P67 was dosed into
these mice twice a week. Urine was collected on day 6 (post single
mRNA dose) and day 13 (post 3 repeat mRNA doses) following AAV
treatment and analyzed for orotic acid levels that were normalized
to creatinine levels. Significant reduction of orotic acid was seen
following OTC mRNA treatment to near normal levels (see FIG. 1A).
Plasma was collected on day 13 (post 3 repeat mRNA doses) following
AAV treatment and analyzed for ammonia levels. Plasma ammonia in
OTC mRNA treated mice were at normal levels similar to that in wild
type and untreated OTC-spf.sup.ash mice compared to hyperammonemic
buffer treated mice (see FIG. 1B).
[0653] In a separate hyperammonemia study in OTC-spf.sup.ash mice
similar to that above, 1 mg/kg of OTC mRNA formulated in
DOTAP:CHEMS:CHOL:DSPE-PEG.sub.2k (50:32:8:10) at N:P 7+co-injection
of 35 mg/kg P82 was dosed into these mice twice a week. Urine was
collected on day 6 (post single mRNA dose) and day 13 (post 3
repeat mRNA doses) following AAV treatment and analyzed for orotic
acid levels that were normalized to creatinine levels. Significant
reduction of orotic acid was seen following OTC mRNA treatment to
normal levels (see FIG. 2A). Plasma was collected on day 13 (post 3
repeat mRNA doses) following AAV treatment and analyzed for ammonia
levels. Plasma ammonia in OTC mRNA treated mice were normalized
compared to hyperammonemic buffer treated mice (see FIG. 2B).
Example 22: Preparation of DSPE-PEG.sub.2K-NAG
##STR00037##
[0655] To compound 3a (204 mg, 0.665 mmol, 2 eq) was added DMF (1.5
mL), and the solution was stirred for 25 min. To the resulting
solution was added trimethylamine (TEA, 185 .mu.L, 1.33 mmol, 4
eq). After 5 min, DSPE-020GS (NOF, 1.00 g, 0.332 mmol, 1 eq) was
added, followed by dichloromethane (DCM, 2.0 mL) and additional DMF
(0.5 mL), and the resulting solution was stirred at ambient
temperature. After 5 h, solvent was removed under reduced
atmosphere, and the residue was taken up in DCM (100 mL). The DCM
layer was washed with saturated NaHCO.sub.3 (30 mL). The resulting
NaHCO.sub.3 layer was washed with DCM (50 mL). The combined organic
layer was dried (Na.sub.2SO.sub.4), and concentrated under reduced
atmosphere. The resulting residue was purified by silica gel
chromatography (2.5.times.7.5 cm, eluent=10% MeOH/DCM (300 mL),
then 15% MeOH/DCM (400 mL), then 20% MeOH/DCM (600 mL), fraction
size=18.times.150 mm test tubes, fractions collected after 125 mL
eluent eluded from column). Fractions 11-40 were concentrated under
reduced atmosphere to afford DSPE-PEG.sub.2K-NAG (439 mg, 41%
yield).
Example 23: In Vivo Expression of mRNA with Repeat Doses of
DOTAP:CHEMS:Cholesterol:DMPE-PEG2k and
DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k mRNA Formulations and
Co-Injection of Polymer
[0656] LNP formulations co-injected with polymer were tested for
mRNA expression using a repeat dosing regime. Co-injections of
mRNA/LNP+polymer and evaluation of in vivo luciferase expression
were performed using the same methods as described in Example
2.
[0657] Table 90 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DMPE-PEG2k or
DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc mRNA nanoparticles with
co-injection of polymer P103. mRNA/LNP+polymer were mixed at a 1:1
ratio and injected immediately into mice. Data was acquired at 6
hours post each dose. Formulations were repeat dosed by IV
administration once a week for 10 weeks in CD-1 mice. Repeat
administration with LNP containing an exchangeable PEG lipid,
DMPE-PEG2K, resulted in similar luminescent signal at each weekly
dose out to 10 weeks. In contrast, repeat administration with LNP
containing a stable PEG lipid, DSPE-PEG2K, resulted in a
significant 20-fold drop in activity starting at week 3. This
decrease ranged from 4 to 30-fold drop in activity over the
subsequent 8 repeat doses compared to week 1 activity.
TABLE-US-00092 TABLE 90 Fluc 2 Fold mRNA Repeat Total Flux
Reduction Lipid-mRNA Dose dosing (photons/sec) from Week 1
Nanoparticle (mg/kg) Polymer time point Geomean STDEV Activity
DOTAP:CHEMS: 0.5 30 mg/kg Week 1 3.07E+10 1.70E+10 1 CHOL:DMPE-
P103 Week 2 3.02E+10 2.79E+10 1.0 PEG2K Week 3 4.35E+10 2.16E+10
0.7 (50:32:16:2) Week 4 1.95E+10 1.16E+10 1.6 N:P 7 13 mg/kg Week 5
8.85E+09 5.78E+09 3.5 Week 6 3.05E+10 1.24E+10 1.0 Week 7 2.57E+10
1.21E+10 1.2 Week 8 1.55E+10 1.07E+10 2.0 Week 9 2.72E+10 1.49E+10
1.1 Week 10 1.41E+10 5.50E+09 2.2 DOTAP:CHEMS: 0.5 30 mg/kg Week 1
1.45E+10 9.30E+09 1 CHOL:DSPE- P103 Week 2 8.83E+09 7.23E+09 1.6
PEG2K Week 3 7.03E+08 1.15E+09 20.6 (50:32:8:10) Week 4 7.49E+08
7.48E+08 19.4 N:P 7 17 mg/kg Week 5 4.72E+08 3.54E+08 30.7 Week 6
3.39E+09 3.53E+09 4.3 Week 7 9.52E+08 9.55E+08 15.2 Week 8 1.39E+09
1.16E+09 10.4 Week 9 2.67E+09 2.32E+09 5.4 Week 10 1.75E+09
1.89E+09 8.3
Example 24: Treatment of Argininosuccinic Aciduria with mRNA
Formulations in a Hypomorphic Argininosuccinic Lyase (ASL) Mouse
Model
[0658] Groups of 5-10 hypomorphic Asl.sup.Neo/Neo mice are treated
by intravenous route of administration with mRNA encoding
argininosuccinic lyase (ASL) formulated in a lipid nanoparticle,
either co-injected or sequentially injected with a
membrane-destabilizing polymer that targets hepatocytes in the
liver as described herein, thereby achieving expression and
activity of ASL. Mice are treated with vehicle control or Asl mRNA
from 0.1-5 mg/kg. Either single or repeat dosing is performed with
a variety of dosing intervals (e.g., daily, every 2 days, biweekly,
etc.). Blood is collected to examine plasma amino acids
(argininosuccinic acid, citrulline, arginine), plasma ammonia, and
serum transaminases at different time points ranging from 3 hours
to 72 hours post final dose on the short term or up to 2 weeks post
dose for duration of effect. At these time points, mice are
sacrificed and livers collected and sampled to measure ASL enzyme
activity, ASL protein expression by western analysis and
immunofluorescence of liver tissue sections. Body weights are
measured if longer term studies are carried out to monitor growth
and survival as the Asl.sup.Neo/Neo mice have significant growth
restrictions and mice die within 6 to 14 weeks of life despite
ongoing treatment with triple therapy (sodium benzoate, sodium
nitrite, L-arginine) (Erez et al., Nat Med 2011. 17:1619-1626).
[0659] Results are compared to vehicle-treated mice as well as to
wild-type littermate mice that have normal levels of ASL protein
expression, plasma amino acid levels, plasma ammonia, and serum
transaminases. Efficacy is shown by detectable levels of ASL
protein expression evaluated by western and immunofluorescence that
is above the level detected in vehicle treated mice. Plasma
argininosuccinc acid (ASA) levels are normally not detectable and
plasma citrulline levels are .about.70 .mu.M in wild-type
littermate mice whereas Asl.sup.Neo/Neo mice have .about.100 .mu.M
ASA and .about.200 .mu.M citrulline levels. Plasma ammonia levels
in wild-type littermate mice are normal, .about.50 .mu.M, whereas
in Asl.sup.Neo/Neo mice levels are elevated in the range of 100-500
.mu.M. Efficacy by plasma amino acid and plasma ammonia levels is a
correction towards levels seen in wild-type littermate mice. In
longer term studies efficacy is shown by increased growth and
survival in comparison to vehicle treated mice.
Example 25: Treatment of Citrullinemia Type 1 (CTLN1) with mRNA
Formulations in a Argininosuccinic Synthetase (ASS1) Deficient
Murine Model of CTLN1 (Fold/Fold)
[0660] Groups of 5-10 Ass1.sup.fold/fold mice are treated by
intravenous route of administration with mRNA encoding
argininosuccinic synthetase (ASS1) formulated in a lipid
nanoparticle, either co-injected or sequentially injected with a
membrane-destabilizing polymer that targets hepatocytes in the
liver as described herein, thereby achieving expression and
activity of ASS1. Mice are treated with vehicle control or Ass1
mRNA from 0.1-5 mg/kg. Either single or repeat dosing is performed
with a variety of dosing intervals (e.g., daily, every 2 days,
biweekly, etc.). Blood is collected to examine plasma amino acids
(citrulline, arginine) and plasma ammonia levels at different time
points ranging from 3 hours to 72 hours post final dose on the
short term or up to 2 weeks post dose for duration of effect. At
these time points, mice are sacrificed and livers collected and
sampled to measure ASS1 enzyme activity, ASS1 protein expression by
western analysis and immunofluorescence of liver tissue sections.
Body weights are measured if longer term studies are carried out to
monitor growth and survival as the Ass1.sup.fold/fold mice have
growth restrictions and die within the first 3 weeks of life if not
treated with sodium benzoate and L-arginine (Perez et al., Am J
Pathol. 177:1958-1968, 2010).
[0661] Results are compared to vehicle-treated mice as well as to
wild-type littermate mice that have normal levels of ASS1 enzyme
activity, plasma amino acid and plasma ammonia levels. Efficacy is
shown by correction of ASS1 enzyme activity that is above the level
detected in vehicle treated mice. Plasma citrulline levels are
.about.70 .mu.M in wild-type littermate mice whereas
Ass1.sup.fold/fold mice have significantly elevated citrulline
levels, .about.2000-3000 .mu.M. Plasma ammonia levels in wild-type
littermate mice are normal, .about.50 .mu.M, whereas
Ass1.sup.fold/fold mice have elevations in the range of 100-500
.mu.M. Levels are high if mice are not treated with sodium benzoate
and L-arginine. Efficacy by plasma amino acid and plasma ammonia
levels is a correction towards levels seen in wild-type littermate
mice. In longer term studies efficacy is shown by increased growth
and survival in comparison to vehicle treated mice if mice are
taken off sodium benzoate and L-arginine treatment.
Example 26: DOTAPen:CHEMS:Cholesterol:DMPE-PEG.sub.2k mRNA
Nanoparticle Formulation with Sequential or Co-Injection of a
Polymer: Formulation Characteristics
[0662] (R)--N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium
chloride (DOTAPen) was synthesized as described in Example 34 and
solubilized at 50 mg/mL in 200 proof ethanol at room temperature
for 15 minutes. The DMPE-PEG.sub.2K (Corden Pharma, Boulder, Colo.,
USA; catalog number LP-R4-123) was solubilized at 50 mg/mL in 200
proof ethanol at room temperature for 15 minutes. The cholesteryl
hemisuccinate (CHEMS) (Avanti Polar Lipid Alabaster, Ala., USA;
catalog number 850524P) and the Cholesterol (CHOL) (Corden Pharma,
Boulder, Colo., USA; catalog number CH-0355) were individually
solubilized at 25 mg/mL in 200 proof at 75.degree. C. for 5
minutes. For a 2 mL preparation of
DOTAPen:CHEMS:CHOL:DMPE-PEG.sub.2K (50:32:16:2 mol %) LNP at a N:P
ratio of 7, a lipid ethanolic mixture containing 92 .mu.L of
DOTAPen at 50 mg/mL in 200 proof ethanol, 79 .mu.L of CHEMS at 25
mg/mL in 200 proof ethanol, 32 .mu.L of CHOL at 25 mg/mL in 200
proof ethanol, 14 .mu.L of DMPE-PEG.sub.2K at 50 mg/mL in 200 proof
ethanol and 450 .mu.L of 200 proof ethanol was prepared for a final
volume of 0.666 mL and a total lipid concentration of 27 mg/mL.
[0663] The lipid nanoparticle (LNP) formulations were prepared at
N:P (nitrogen to phosphate) ratios from 7 to 10 based on the
DOTAPen concentration. The DOTAPen:CHEMS ratio was fixed at 1.6 at
50:32 mol % respectively at the various N:P ratios.
[0664] The Fluc (firefly luciferase) mRNA stock solution at 1 mg/mL
in 10 mM Tris-HCl (pH 7.5) was diluted to 0.225 mg/mL in 300 mM
sucrose 20 mM phosphate, pH 7.4 buffer (SUP buffer). The mRNA/LNPs
were assembled at N:P ratios from 7 or 10 by mixing the ethanolic
lipid solution with 0.225 mg/mL mRNA in SUP buffer at a 1:2 ratio
(lipid ethanolic mixture:mRNA in SUP buffer) using the microfluidic
device from Precision NanoSystems Inc (Vancouver BC, Canada) at a
12 mL/minute flow rate. The mRNA/LNPs in 33% ethanol were then
incubated at room temperature for 60 minutes prior to dialysis for
18 hours against 100 volumes (200 mL) of SUP buffer.
[0665] The polymers used for co-injection were solubilized at 20
mg/mL in SUP buffer with agitation at 400 rpm for 1 hour and then
stored overnight at 4.degree. C. The polymers were diluted to 6
mg/mL in SUP buffer prior to injection.
[0666] Since the mRNA/LNP and polymer were co-injected, a 2.times.
solution of each was prepared. Just prior to dosing, the solutions
were mixed and injected immediately.
[0667] The formulation particle size was measured by adding 10
.mu.L of formulation to 90 .mu.L of SUP buffer into a disposable
micro-cuvette and analyzed using the Malvern Instrument ZETASIZER
NANO-ZS. The LNPs showed a particle size of 88 nm (Z-average). The
formulation zeta-potential at pH 7.4 was measured by adding 10
.mu.L of formulation to 740 .mu.L of SUP buffer into a disposable 1
mL cuvette. The formulation zeta-potential at pH 4 was measured by
adding 10 .mu.L of formulation to 740 .mu.L of sucrose acetate
buffer (pH 4) into a disposable 1 mL cuvette. The zeta dip cell was
inserted into the 1 mL cuvette and the formulation was analyzed
using the ZETASIZER NANO-ZS. The DOTAPen LNPs had a zeta potential
of -4 mV at pH 7 and +12 mV at pH 4. The ability of the LNP to
compact the mRNA was measured in a 96-well plate using a RiboGreen
dye accessibility assay. 100 .mu.L of nanoparticles diluted 1:64 in
SUP for the dye accessible mRNA measurement or 100 .mu.L of
nanoparticles diluted 1:200 in SUP for total mRNA measurement was
loaded in a 96-well plate. To this, 100 .mu.L of a 1:200 dilution
of RiboGreen reagent in SUP buffer for the dye accessible
measurement or 100 .mu.L of a 1:200 dilution of RiboGreen reagent
in 0.2% Triton X-100/SUP buffer for the total mRNA measurement, was
added to each well, respectively. The plate was incubated at room
temperature in the dark for 5 minutes. The fluorescence was read
using a Molecular Devices SpectraMax M5 with excitation at 480 nm
and emission at 520 nm. Finally, the percent dye accessibility was
calculated by subtracting the .mu.M concentration of dye accessible
mRNA from the .mu.M concentration of the total mRNA, dividing that
value by the .mu.M concentration of total mRNA, and then
multiplying by 100.
[0668] The DOTAPen LNPs showed 28% dye accessibility when prepared
in SUP buffer. Table 91 below shows characterization of exemplary
LNP formulations.
TABLE-US-00093 TABLE 91 LNPs Characteristics Sample # RP659-1
RP659-2 Lipid DOTAPen:CHEMS:CHOL: DMPE-PEG2K (50:32:16:2) N/P 10 7
Lipid Concentration (mg/mL) 3.9 2.7 Visual Appearance Opalescent
(+) Opalescent (+) % Dye access SUP pH 7.4 50% 28% Z-Ave (nm) 83 88
PDI 0.051 0.070 Number (nm) 64 63 Pk 1 Mean Int (nm) 88 95 Pk 2
Mean Int (nm) 0 0 Pk 1 Area Int (%) 100 100 Pk 2 Area Int (%) 0 0
ZP pH 7.4 (mV) -6 -4 ZP pH 4 (mV) 10 12 Sizing data quality GOOD
GOOD
Example 27: In Vivo Expression of mRNA with
DOTAPen:CHEMS:Cholesterol:DMPE-PEG.sub.2k mRNA Formulations and
Co-Injection of Polymer
[0669] DOTAPen-containing LNPs described in Example 26 were tested
with P105 using co-injection and the same methods as described in
Example 2.
[0670] Table 92 displays luminescence values in the liver for
animals treated with DOTAPen:CHEMS:CHOL:DMPE-PEG2k+Fluc mRNA
nanoparticles at N:P ratio of 7 or 10 with co-injection of polymer
P105. Activity of DOTAPen-containing LNPs was compared to
DOTAP:CHEMS:CHOL:DSPE-PEG2k+Fluc mRNA nanoparticles with
co-injection of polymer. mRNA/LNP+polymer were mixed at a 1:1 ratio
and injected immediately into mice. Data was acquired at 6 hours
post dose. Fluc mRNA/DOTAPen:CHEMS:CHOL:DSPE-PEG2k LNP+P105 showed
3 to 6-fold lower luminescent signal compared to Fluc
mRNA/DOTAP:CHEMS:CHOL:DSPE-PEG2k LNP+P105.
TABLE-US-00094 TABLE 92 Fluc 2 Polymer Total Flux Lipid-mRNA mRNA
Dose Dose (photons/sec) Nanoparticle (mg/kg) Polymer (mg/kg)
Geomean STDEV DOTAP:CHEMS:CHOL: 0.5 P105 30 1.80E+10 1.00E+10
DSPE-PEG2K (50:32:8:10) N:P 7 17.5 mg/kg DOTAPen:CHEMS:CHOL: 0.5
P105 30 5.16E+09 5.74E+09 DMPE-PEG2K (50:32:16:2) N:P 10 17.5 mg/kg
DOTAPen:CHEMS:CHOL: 0.5 P105 30 2.93E+09 3.45E+09 DMPE-PEG2K
(50:32:16:2) N:P 7 17.5 mg/kg
Example 28: In Vivo Expression of hEPO mRNA with
DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k mRNA Formulations and
Co-Injection of Polymer
[0671] Female CD-1 mice (7-10 weeks old) were used for evaluating
hEPO mRNA formulated in DOTAP:CHEMS:Cholesterol:DSPE-PEG2k LNP with
co-injection of P96 polymer. The formulation was dosed
intravenously at 1 mg/kg of mRNA, 35 mg/kg of lipid, and 35 mg/kg
of polymer with 5 mice injected per group. Mice injected with
sucrose phosphate buffer were used as control. For each injection
mice were given a final dose volume of approximately 0.25 mL or 10
mL/kg based on individual body weights.
[0672] The in vivo expression of hEPO mRNA was evaluated in mouse
serum collected at 6 hours post dose. Blood was taken by
retro-orbital sampling and collected in serum separator tubes.
Serum was isolated by centrifugation and stored frozen at
-20.degree. C. until assayed. For ELISA assay the serum was diluted
in PBS and then run using Human Epo Quantikine IVD ELISA (R&D
Systems #DEPOO) according to manufacturer's protocol. Briefly, 100
.mu.L of diluted sample was mixed with 100 .mu.L Epo assay diluent
in an ELISA plate and shaken at 500 RPM for 1 hour. The solution
was removed and replaced with 200 .mu.L of antibody conjugate and
shaken for an additional hour. The plate was then washed and
developed using a two component HRP/TMB system and read at 450
nm.
[0673] Table 93 displays hEPO serum levels for animals treated with
buffer or with hEPO mRNA/LNP with co-injection of polymer P96. No
detectable levels of hEPO were seen in buffer treated mice in
comparison to 2.98.times.10.sup.6 .mu.g/mL of hEPO detected with 1
mg/kg of hEPO mRNA.
TABLE-US-00095 TABLE 93 hEPO Polymer hEPO serum levels Lipid-mRNA
mRNA Dose Dose (pg/mL) Nanoparticle (mg/kg) Polymer (mg/kg) Average
STDEV None 0 none none <2.5 DOTAP:CHEMS:CHOL: 1 P96 35 2.98E+06
1.24E+06 DSPE-PEG2K (50:32:8:10) N:P 7 35 mg/kg
Example 29: In Vivo Cytokine Analysis of HPLC-Purified and
Non-Purified mRNAs with DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k
mRNA Formulations and Co-Injection of Polymer
[0674] Female CD-1 mice (7-10 weeks old) were used for evaluating
HPLC-purified or non-purified Fluc mRNA formulated in
DOTAP:CHEMS:Cholesterol:DSPE-PEG2k LNP with co-injection of P95
polymer. The formulation was dosed intravenously at 1 mg/kg of
mRNA, 35 mg/kg of lipid, and 30 mg/kg of polymer with 5 mice
injected per group. Mice injected with sucrose phosphate buffer
were used as control. For each injection mice were given a final
dose volume of approximately 0.25 mL or 10 mL/kg based on
individual body weights.
[0675] Mouse IP-10 cytokine levels were quantified using R&D
systems Mouse CXCL10/IP-10/CRG-2 Quantikine ELISA kit (#SMCX100).
Blood was taken by retro-orbital sampling at 3 hours post dose and
collected in serum separator tubes. Serum was isolated by
centrifugation and stored frozen at -20.degree. C. until assayed.
For ELISA the serum was diluted in PBS and then run according to
manufacturer's protocol. Briefly, 50 .mu.L of diluted sample was
mixed with 50 .mu.L assay diluent in an ELISA plate and incubated
at RT for two hours. The solution was removed and replaced with 200
.mu.L of antibody conjugate and incubated at RT for two hours. The
plate was then washed and developed using a two component HRP/TMB
system and read at 450 nm.
[0676] Table 94 displays IP-10 serum levels for animals treated
with buffer or with HPLC-purified or non-purified Fluc mRNA
formulated in LNP with co-injection of polymer P95. IP-10 cytokine
levels at 3 hours post dose were significantly reduced with
HPLC-purified Fluc mRNA in comparison to high IP-10 cytokine levels
induced with non-purified Fluc mRNA.
TABLE-US-00096 TABLE 94 Polymer Mouse IP-10 serum Lipid-mRNA Dose
levels (pg/mL) Nanoparticle mRNA Polymer (mg/kg) Average STDEV None
0 none none <30 DOTAP:CHEMS:CHOL: 0.5 mg/kg Non- P95 30 10293
4524 DSPE-PEG2K Purified Fluc 2 (50:32:16:2) mRNA N:P 7 17 or 35
mg/kg 1 mg/kg Non- P95 30 14827 2824 Purified Fluc 2 mRNA 0.5 mg/kg
HPLC- P95 30 644 639 Purified Fluc 2 mRNA 1 mg/kg HPLC- P95 30 2377
3175 Purified Fluc 2 mRNA
Example 30: In Vivo Expression of HPLC-Purified or Non-Purified
Flue mRNA with DOTAP:CHEMS:Cholesterol:DSPE-PEG.sub.2k and
Co-Injection of Polymer Following Repeat Dosing
[0677] HPLC-purified Fluc 2 mRNA and non-purified Fluc 2 mRNA
formulated in DOTAP:CHEMS:CHOL:DSPE-PEG2k LNPs with P95 using
co-injection were repeat dosed in CD-1 mice using the same methods
described in Example 2.
[0678] Table 95 displays luminescence values in the liver for
animals treated with DOTAP:CHEMS:CHOL:DSPE-PEG2k+HPLC-purified or
non-purified Fluc 2 mRNA nanoparticles with co-injection of polymer
P95. mRNA/LNP+polymer were mixed at a 1:1 ratio and injected
immediately into mice. Data was acquired at 6 hours post each dose.
Formulations were repeat dosed by IV administration once a week for
5 weeks in CD-1 mice. Repeat administration with HPLC-purified Fluc
mRNA resulted in little reduction in luminescent signal (up to
8-fold) at each weekly dose out to 5 weeks. In contrast, repeat
administration with non-purified Fluc mRNA resulted in up to
76-fold reduction in luminescent signal at each weekly dose out to
5 weeks.
TABLE-US-00097 TABLE 95 Fold Repeat Total Flux Reduction Lipid-mRNA
mRNA dosing (photons/sec) from Week 1 Nanoparticle Dose Polymer
time point Geomean STDEV Activity DOTAP:CHEMS: 0.5 mg/kg of 30
mg/kg Week 1 8.02E+09 5.83E+09 1 CHOL:DMPE- Non-Purified P95 Week 2
1.50E+09 3.16E+09 5.3 PEG2K Fluc 2 mRNA Week 3 1.79E+08 2.32E+08
44.9 (50:32:16:2) Week 4 1.05E+08 4.96E+07 76.6 N:P 7 17.5 mg/kg
Week 5 3.10E+08 9.54E+08 25.9 0.5 mg/kg of 30 mg/kg Week 1 1.09E+10
1.12E+10 1 HPLC-Purified P95 Week 2 4.82E+09 2.03E+09 2.3 Fluc2
mRNA Week 3 1.30E+09 8.92E+09 8.4 Week 4 1.82E+09 4.61E+09 6.0 Week
5 5.29E+09 1.20E+10 2.1
Example 31: In Vivo Cytokine Analysis of HPLC-Purified and
Non-Purified mRNAs with DOTAP:CHEMS:Cholesterol:DMPE-PEG.sub.2k
mRNA Formulations and Co-Injection of Polymer
[0679] Male OTC-spf.sup.ash mice (8-12 weeks old) were used for
evaluating HPLC purified or non-purified hOTC or untranslatable
hOTC control mRNA (AUG start codon was mutated to AAG) formulated
in DOTAP:CHEMS:Cholesterol:DMPE-PEG2k LNP with co-injection of P103
polymer. The formulation was dosed intravenously at 1 mg/kg of
mRNA, 27 mg/kg of lipid, and 30 mg/kg of polymer with 5 mice
injected per group. Mice injected with sucrose phosphate buffer
were used as control. For each injection mice were given a final
dose volume of approximately 0.25 mL or 10 mL/kg based on
individual body weights.
[0680] Mouse IP-10 cytokine levels were quantified using R&D
systems Mouse CXCL10/IP-10/CRG-2 Quantikine ELISA kit (#SMCX100).
Blood was taken by retro-orbital sampling at 3 hours post dose and
collected in serum separator tubes. Serum was isolated by
centrifugation and stored frozen at -20.degree. C. until assayed.
For ELISA the serum was diluted in PBS and then run according to
manufacturer's protocol. Briefly, 50 .mu.L of diluted sample was
mixed with 50 .mu.L assay diluent in an ELISA plate and incubated
at RT for two hours. The solution was removed and replaced with 200
.mu.L of antibody conjugate and incubated at RT for two hours. The
plate was then washed and developed using a two component HRP/TMB
system and read at 450 nm.
[0681] Table 96 displays IP-10 serum levels for animals treated
with buffer or with HPLC-purified or non-purified hOTC mRNA or
untranslatable hOTC control mRNA formulated in LNP with
co-injection of polymer P103. No induction of IP-10 cytokine levels
at 3 hours post dose was observed with HPLC-purified mRNA in
comparison to high IP-10 cytokine levels induced with non-purified
mRNA.
TABLE-US-00098 TABLE 96 Polymer Mouse IP-10 serum Lipid-mRNA Dose
levels (pg/mL) Nanoparticle mRNA Polymer (mg/kg) Average STDEV None
- Buffer 0 none none <30 DOTAP:CHEMS:CHOL: 1 mg/kg HPLC-Purified
P103 30 <30 DMPE-PEG2K hOTC (50:32:16:2) 1 mg/kg Non-Purified
P103 30 8337 506 N:P 7 27 mg/kg hOTC 1 mg/kg HPLC-Purified P103 30
<30 untranslatable hOTC control 1 mg/kg Non-Purified P103 30
5622 1330 untranslatable hOTC control
Example 32: Therapeutic Efficacy of HPLC-Purified mRNA with
Lipid-mRNA Formulations and Co-Injection of Polymer in Ornithine
Transcarbamylase Deficient Mice
[0682] Hyperammonemia was induced in OTC-spf.sup.ash mice as
described in Example 21. Four (4) days after AAV dosing, 1 mg/kg of
HPLC-purified OTC mRNA or 1 mg/kg of HPLC-purified untranslatable
OTC control mRNA formulated in
DOTAP:CHEMS:CHOL:DMPE-PEG.sub.2k(50:32:16:2) at N:P 7+co-injection
of 30 mg/kg polymer P103 was administered every 3 to 4 days for a
total of 3 repeat doses. Urine was collected 48 h post the second
mRNA dose (on day 9 following AAV treatment) and analyzed for
orotic acid levels that were normalized to creatinine levels.
Orotic acid (OA) levels were reduced following OTC mRNA treatment
(336.+-.166 .mu.mol OA/mmol creatinine) in comparison to buffer
treatment (999.+-.192 .mu.mol OA/mmol creatinine) or untranslatable
control mRNA treatment (882.+-.192 .mu.mol OA/mmol creatinine).
Plasma was collected on day 12 (24 h post 3rd repeat mRNA dose)
following AAV treatment and analyzed for ammonia levels. Plasma
ammonia levels were reduced to normal levels (43.+-.29 .mu.M
ammonia) following treatment with OTC mRNA in comparison to
hyperammonemic mice treated with untranslatable control mRNA
(217.+-.119 .mu.M ammonia) or buffer treatment (110.+-.24 .mu.M
ammonia). To examine whether any cytokine induction was observed
following administration of HPLC-purified OTC or untranslatable
control mRNA, serum was collected at 3 h post the first mRNA dose
and examined for IP-10 levels. IP-10 levels were below the level of
quantitation (<30 .mu.g/mL) in both HPLC-purified OTC mRNA and
untranslatable control mRNA treated mice, similar to buffer treated
mice. In contrast, unpurified Fluc 2 mRNA control showed high
induction of IP-10 serum levels (13,009.+-.4932 .mu.g/mL).
Example 33: Expression of OTC mRNA with Lipid-mRNA Formulations and
Co-Injection of Polymer in Ornithine Transcarbamylase Deficient
Mice
[0683] OTC-spf.sup.ash mice were administered a single IV dose of 3
mg/kg of OTC mRNA, 3 mg/kg of untranslatable OTC control mRNA, or
buffer. Each mRNA was formulated in
DOTAP:CHEMS:CHOL:DSPE-PEG.sub.2k (50:32:18:10) at N:P
7+co-injection of 30 mg/kg polymer P105. Mice were sacrificed at 6,
24, or 48 h post dose and liver tissue samples were collected for
OTC western analysis. To prepare protein extracts from liver
tissue, 400-600 .mu.l of freshly prepared Pierce T-PER tissue lysis
buffer (1 Pierce protease and phosphatase inhibitor cocktail tablet
for 10 ml of lysis buffer) was added into each sample tube
containing approximately 200 mg of liver tissue. Tubes were then
loaded onto MP Bio Fastprep-24 Instrument (Cat#116004500) to
homogenize tissue for 20 seconds at a speed of 6 m/s. Each tissue
homogenate was centrifuged at 4.degree. C., 13,000 rpm for 15
minutes, and the supernatant was transferred to a new Eppendorf
tube. This whole cell lysate was further analyzed for total protein
concentration by BCA assay (Thermo Scientific, Cat#23225). 25 .mu.g
of each sample was loaded per lane on 4-12% SDS-PAGE gels (Bio-Rad,
Cat#345-0124) after mixing protein extract with 4.times. sample
buffer (Bio-Rad, Cat#161-0791) and 20.times.XT Reducing Reagent
(Bio-Rad, Cat#161-0792) for a final protein concentration of 5
.mu.g/l. Samples were then heated at 95.degree. C. for 5 minutes
prior to running on gel. Following electrophoresis, blotting was
performed by transferring proteins from gels to PVDF membranes
(Bio-Rad, Cat#170-4157) under Bio-Rad Transfer-Blot Turbo system
(Cat#170-4155). Subsequently, the blots were blocked in Odyssey
Blocking Buffer (LI-COR, Cat#927-40000) at room temperature for 1
hour, followed by incubation with OTC (Sigma, Cat# HPA000243,
1:2000 dilution) or HSP90 (Origene, Cat#TA500494, 1:8000 dilution)
primary antibodies at 4.degree. C. overnight. After several washes
in TBST buffer, the blots were incubated with HRP-conjugated
secondary antibody (Cell Signaling, Cat#7076S, 1:2000) at room
temperature for 1 hour. To visualize protein bands, the washed
blots were incubated with luminescence-based HRP substrate
(Millipore, Cat# WBLUF0500) and then imaged under Bio-Rad ChemiDoc
XRS system (Cat#170-8265). The quantification of westerns was
performed using Bio-Rad Image Lab Software (Cat#170-9690) linked to
the ChemiDoc system. To quantitate OTC expression levels in treated
OTC-spf.sup.ash samples relative to wild-type littermate sample,
the intensity of the OTC protein band was divided by that of
loading control HSP90 in the same sample. This ratio was then
divided by a similar ratio of a wild-type littermate sample. This
was viewed as % OTC expression relative to wild-type.
[0684] Table 97 displays % OTC expression relative to wild-type
littermate mouse for OTC-spf.sup.ash mice treated with 3 mg/kg of
OTC mRNA, 3 mg/kg untranslatable control mRNA, or buffer. At 24 and
48 h post dose, OTC mRNA treatment in OTC-spf.sup.ash mice showed
approximately 40% of wild-type OTC expression levels. No OTC
expression was detectable from untranslatable control mRNA above
the level seen with buffer treatment.
TABLE-US-00099 TABLE 97 Polymer Time % OTC Expression Lipid-mRNA
Dose Point Relative to Wild-Type Nanoparticle Treatment (mg/kg) (h)
AVG STDEV DOTAP:CHEMS:CHOL: Buffer none 6 h 10.4% 2% DSPE-PEG2K 3
mg/kg OTC 30 mg/kg 6 h 13.4% 4.2% (50:32:8:10) mRNA N:P 7 105 mg/kg
24 h 41.1% 13.1% 48 h 41.6% 10.1% 3 mg/kg 30 mg/kg 6 h 10.1% 1%
untranslatable control mRNA 24 h 9.9% 1.2%
Example 34: Synthesis of Cationic Lipids
Part 1: Synthesis of (R)-5-(dimethylamino)pentane-1,2-diyl dioleate
hydrochloride (DODAPen-CI)
##STR00038##
[0686] (R)-5-bromopentane-1,2-diyl dioleate (1.66 g, 2.33 mmol) was
dissolved in anhydrous acetonitrile (50.0 mL) in a 100 mL round
bottom flask equipped with a magnetic stirring bar. Dimethylamine
hydrochloride (0.951 g, 11.7 mmol) and diisopropylethylamine (2.03
mL, 11.7 mmol) were added successively to the suspension and the
mixture was heated to 60.degree. C. in an oil bath for 16 h. The
now-clear solution was cooled to RT (upon which it turned cloudy)
and the solvent was removed under reduced pressure on the rotovap
to afford a brown oily residue. The crude residue was purified by
silica gel chromatography using a gradient of dichloromethane:
methanol (0 to 10%) to afford the clean product as a light brown
semi-solid (1.20 g, 1.77 mmol. Yield: 76%). The hydrochloride salt
was obtained by adding concentrated hydrochloric acid to the oily
product and concentrating the mixture to dryness on the rotovap and
subsequently under high vacuum. The final product was obtained as a
waxy off-white. The final product was characterized by NMR (400 MHz
1H NMR with CD.sub.3OD as solvent) and all spectra were consistent
with the desired.
Part 2: Synthesis of (R)-5-guanidinopentane-1,2-diyl dioleate
hydrochloride (DOPen-G)
##STR00039##
[0687] Part 2A: Synthesis of
(R)-5-((tert-butoxycarbonyl)amino)pentane-1,2-diyl dioleate
[0688] (R)-tert-butyl-(4,5-dihydroxypentyl)carbamate (2.10 g, 9.58
mmol) was dissolved in anhydrous dichloromethane (50.0 mL) in a 250
mL round bottom flask equipped with a magnetic stirring bar. Oleic
acid (5.70 g, 20.2 mmol) was added to the mixture and the stirring
solution was cooled to 0.degree. C. in an ice bath.
Dicyclohexylcarbodiimide (4.94 g, 23.9 mmol) and
dimethylaminopyridine (1.17 g, 9.58 mmol) were added to the cold
solution and the reaction was warmed to RT over 16 h. The solid
dicyclohexyl urea precipitate was filtered out on a Buchner funnel
and washed with dichloromethane (4.times.25 mL). The
dichloromethane filtrate was concentrated under reduced pressure on
a rotovap to obtain an oily residue. The resulting residue was
purified by silica gel chromatography using a gradient of
hexane:ethyl acetate (0 to 10%). The pure product was obtained as a
colorless oil (6.89 g, 9.21 mmol). Yield: 96%. The product was
characterized by NMR (400 MHz 1H NMR with CD.sub.3OD as solvent)
and all spectra were consistent with
(R)-5-((tert-butoxycarbonyl)amino)pentane-1,2-diyl dioleate.
Part 2B: Synthesis of (R)-5-aminopentane-1,2-diyl dioleate
hydrochloride
[0689] (R)-5-((tert-butoxycarbonyl)amino)pentane-1,2-diyl dioleate
(6.87 g, 9.18 mmol) was dissolved in anhydrous 1,4-dioxane (50.0
mL) in a 250 mL round bottom flask equipped with a magnetic
stirring bar. 4N hydrochloric acid in 1,4-dioxane was added (46.0
mL, 184 mmol) and the solution was stirred at RT for 4 h. The
solvent was removed under reduced pressure on a rotovap and the
product was dried under high vacuum for 16 h. The pure product was
obtained as a viscous colorless oil (6.29 g, 9.18 mmol) in
quantitative yield. The product was characterized by NMR (400 MHz
1H NMR with CD.sub.3OD as solvent) and all spectra were consistent
with (R)-5-aminopentane-1,2-diyl dioleate hydrochloride.
Part 2C: Synthesis of (R)-5-(2,3-bis(tert-butoxycarbonyl)
guanidino)pentane-1,2-diyl dioleate
[0690] (R)-5-aminopentane-1,2-diyl dioleate hydrochloride (2.46 g,
3.59 mmol) was dissolved in anhydrous dichloromethane (50.0 mL) in
a 250 mL round bottom flask equipped with a magnetic stirring bar.
Triethylamine (1.00 mL, 7.17 mmol) and
1,3-Di-Boc-2-(trifluoromethylsulfonyl)guanidine (1.55 g, 3.96 mmol)
were added successively and the mixture was stirred at ambient
temperature for 22 h. The solution was concentrated under reduced
pressure on a rotovap to afford an oily residue. The resulting
residue was purified by silica gel chromatography using a gradient
of hexane:ethyl acetate (0 to 10%). The pure product was obtained
as a colorless oil (3.00 g, 3.37 mmol). Yield: 94%. The product was
characterized by NMR (400 MHz 1H NMR with CDCl.sub.3 as solvent)
and all spectra were consistent with
(R)-5-(2,3-bis(tert-butoxycarbonyl)guanidino)pentane-1,2-diyl
dioleate.
Part D: Synthesis of (R)-5-guanidinopentane-1,2-diyl dioleate
hydrochloride (DOPen-G)
[0691]
(R)-5-(2,3-bis(tert-butoxycarbonyl)guanidino)pentane-1,2-diyl
dioleate (1.81 g, 2.03 mmol) was dissolved in anhydrous 1,4-dioxane
(20.0 mL) in a 250 mL round bottom flask equipped with a magnetic
stirring bar. 4N hydrochloric acid in 1,4-dioxane was added (30.2
mL, 121 mmol) and the solution was stirred at RT for 48 h. The
solvent was removed under reduced pressure on a rotovap to afford
an oily residue. The resulting residue was purified on silica gel
chromatography using a gradient of dichloromethane:methanol (0t
o100%). The pure product was dried under high vacuum for 20 h to
yield an off-white semi-solid (1.00 g, 1.38 mmol). Yield: 68%. The
product was characterized by NMR (400 MHz 1H NMR with CD.sub.3OD as
solvent) and all spectra were consistent with DOPen-G.
Part 3: Synthesis of
(R)--N,N,N-trimethyl-4,5-bis(oleoyloxy)pentan-1-aminium chloride
(DOTAPen)
##STR00040##
[0693] (R)-5-(dimethylamino)pentane-1,2-diyl dioleate (DODAPen,
0.700 g, 1.04 mmol) was dissolved in anhydrous acetonitrile (10.0
mL) in a 100 mL round bottom flask equipped with a magnetic
stirring bar. Diispopropylethylamine (1.80 mL, 10.3 mmol) and
iodomethane (1.93 mL, 31.0 mmol) were added successively and the
solution was refluxed at 85.degree. C. for 20 h. The solution was
cooled to RT and diluted with diethyl ether (300 mL) upon which a
precipitate of diisopropylethylaminium iodide salt formed. The
solid precipitate was filtered out and the combined organic phase
was concentrated under reduced pressure on a rotovap. The crude
residue was passed through a short silica gel column using a
mixture of dichloromethane and methanol (10%). The pure product
(iodide salt) was obtained as a brown-red semi-solid (780 mg). The
product was then passed through an Amberlite IRA 400 chloride
ion-exchange resin column and eluted with a mixture of
dichloromethane:methanol (33%). The column procedure was repeated
10 times to obtain the desired product as the chloride salt. After
drying under high vacuum, the pure product was obtained as a light
brown waxy solid (430 mg, 0.592 mmol). Yield: 57%. The product was
characterized by NMR (400 MHz 1H NMR with CD.sub.3OD as solvent)
and all spectra were consistent with DOTAPen.
[0694] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims. All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entireties for
all purposes.
Sequence CWU 1
1
511354PRTHomo sapiensTRANSIT(1)..(34)native mitochondrial leader
seqeuence 1Met Leu Phe Asn Leu Arg Ile Leu Leu Asn Asn Ala Ala Phe
Arg Asn 1 5 10 15 Gly His Asn Phe Met Val Arg Asn Phe Arg Cys Gly
Gln Pro Leu Gln 20 25 30 Asn Lys Val Gln Leu Lys Gly Arg Asp Leu
Leu Thr Leu Lys Asn Phe 35 40 45 Thr Gly Glu Glu Ile Lys Tyr Met
Leu Trp Leu Ser Ala Asp Leu Lys 50 55 60 Phe Arg Ile Lys Gln Lys
Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys 65 70 75 80 Ser Leu Gly Met
Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser 85 90 95 Thr Glu
Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr 100 105 110
Thr Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr Ala 115
120 125 Arg Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg Val Tyr
Lys 130 135 140 Gln Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala Ser Ile
Pro Ile Ile 145 150 155 160 Asn Gly Leu Ser Asp Leu Tyr His Pro Ile
Gln Ile Leu Ala Asp Tyr 165 170 175 Leu Thr Leu Gln Glu His Tyr Ser
Ser Leu Lys Gly Leu Thr Leu Ser 180 185 190 Trp Ile Gly Asp Gly Asn
Asn Ile Leu His Ser Ile Met Met Ser Ala 195 200 205 Ala Lys Phe Gly
Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu 210 215 220 Pro Asp
Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn 225 230 235
240 Gly Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala His Gly
245 250 255 Gly Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met Gly Gln
Glu Glu 260 265 270 Glu Lys Lys Lys Arg Leu Gln Ala Phe Gln Gly Tyr
Gln Val Thr Met 275 280 285 Lys Thr Ala Lys Val Ala Ala Ser Asp Trp
Thr Phe Leu His Cys Leu 290 295 300 Pro Arg Lys Pro Glu Glu Val Asp
Asp Glu Val Phe Tyr Ser Pro Arg 305 310 315 320 Ser Leu Val Phe Pro
Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala 325 330 335 Val Met Val
Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro 340 345 350 Lys
Phe 2354PRTArtificialHuman ornithine transcarbamylase with mouse
mitochondrial leader sequenceTRANSIT(1)..(34)mouse mitochondrial
leader sequence 2Met Leu Ser Asn Leu Arg Ile Leu Leu Asn Asn Ala
Ala Leu Arg Lys 1 5 10 15 Gly His Thr Ser Val Val Arg His Phe Trp
Cys Gly Lys Pro Val Gln 20 25 30 Ser Gln Val Gln Leu Lys Gly Arg
Asp Leu Leu Thr Leu Lys Asn Phe 35 40 45 Thr Gly Glu Glu Ile Lys
Tyr Met Leu Trp Leu Ser Ala Asp Leu Lys 50 55 60 Phe Arg Ile Lys
Gln Lys Gly Glu Tyr Leu Pro Leu Leu Gln Gly Lys 65 70 75 80 Ser Leu
Gly Met Ile Phe Glu Lys Arg Ser Thr Arg Thr Arg Leu Ser 85 90 95
Thr Glu Thr Gly Phe Ala Leu Leu Gly Gly His Pro Cys Phe Leu Thr 100
105 110 Thr Gln Asp Ile His Leu Gly Val Asn Glu Ser Leu Thr Asp Thr
Ala 115 120 125 Arg Val Leu Ser Ser Met Ala Asp Ala Val Leu Ala Arg
Val Tyr Lys 130 135 140 Gln Ser Asp Leu Asp Thr Leu Ala Lys Glu Ala
Ser Ile Pro Ile Ile 145 150 155 160 Asn Gly Leu Ser Asp Leu Tyr His
Pro Ile Gln Ile Leu Ala Asp Tyr 165 170 175 Leu Thr Leu Gln Glu His
Tyr Ser Ser Leu Lys Gly Leu Thr Leu Ser 180 185 190 Trp Ile Gly Asp
Gly Asn Asn Ile Leu His Ser Ile Met Met Ser Ala 195 200 205 Ala Lys
Phe Gly Met His Leu Gln Ala Ala Thr Pro Lys Gly Tyr Glu 210 215 220
Pro Asp Ala Ser Val Thr Lys Leu Ala Glu Gln Tyr Ala Lys Glu Asn 225
230 235 240 Gly Thr Lys Leu Leu Leu Thr Asn Asp Pro Leu Glu Ala Ala
His Gly 245 250 255 Gly Asn Val Leu Ile Thr Asp Thr Trp Ile Ser Met
Gly Gln Glu Glu 260 265 270 Glu Lys Lys Lys Arg Leu Gln Ala Phe Gln
Gly Tyr Gln Val Thr Met 275 280 285 Lys Thr Ala Lys Val Ala Ala Ser
Asp Trp Thr Phe Leu His Cys Leu 290 295 300 Pro Arg Lys Pro Glu Glu
Val Asp Asp Glu Val Phe Tyr Ser Pro Arg 305 310 315 320 Ser Leu Val
Phe Pro Glu Ala Glu Asn Arg Lys Trp Thr Ile Met Ala 325 330 335 Val
Met Val Ser Leu Leu Thr Asp Tyr Ser Pro Gln Leu Gln Lys Pro 340 345
350 Lys Phe 31262DNAArtificialcDNA encoding human ornithine
transcarbamylase, codon-optimized for mouse
expressionpromoter(1)..(20)T7 promoter 3taatacgact cactataggg
aaataagaga gaaaagaaga gtaagaagaa atataagagc 60caccatgctg ttcaacctca
gaatcctcct caataacgcc gcctttagaa acggtcataa 120cttcatggtc
agaaacttta gatgtggtca gcctctccag aacaaagtgc agctcaaggg
180gcgggacctg ctcaccctga aaaatttcac aggcgaggaa atcaagtaca
tgctctggct 240gtctgccgat ctgaagttca ggatcaagca gaagggcgaa
tatctcccac tgctccaggg 300gaaaagtctg ggtatgatct tcgaaaagcg
gagtactagg accagactgt caacagagac 360tggattcgct ctgctcggag
gacacccatg ctttctgacc acacaggaca ttcatctcgg 420tgtgaacgag
tcactgaccg acacagctcg agtcctcagc tccatggcag atgccgtgct
480ggcaagggtc tacaaacaga gtgacctcga taccctggct aaggaagcaa
gcatccccat 540cattaatgga ctctccgacc tgtatcaccc tatccagatt
ctggccgatt acctcaccct 600gcaggagcat tattctagtc tgaaagggct
cacactgagc tggattggcg acggaaacaa 660tatcctgcac tccattatga
tgtctgccgc taagtttggc atgcatctgc aggcagccac 720accaaaagga
tacgaacccg atgcttccgt gactaagctg gccgaacagt atgctaaaga
780gaacggaact aagctgctcc tgaccaatga ccccctggag gctgcacacg
ggggtaacgt 840cctgatcact gatacctgga tttccatggg ccaggaggaa
gagaagaaaa agcgcctgca 900ggcattccag ggataccagg tgacaatgaa
aactgccaag gtcgccgctt ctgattggac 960ttttctccat tgtctgcccc
gaaagcctga agaggtggac gatgaggtct tctattcacc 1020tcggagcctg
gtgtttccag aagccgagaa tcgcaagtgg acaatcatgg cagtgatggt
1080gtccctcctc acagactatt ccccacagct ccagaagccc aagttttgag
cggccgctta 1140attaagctgc cttctgcggg gcttgccttc tggccatgcc
cttcttctct cccttgcacc 1200tgtacctctt ggtctttgaa taaagcctga
gtaggaagtc tagagtttaa acatttaaat 1260ct 126241262DNAArtificialcDNA
encoding human ornithine transcarbamylase with mouse mitochondrial
leader sequence, codon-optimized for mouse
expressionpromoter(1)..(20)T7 promoter 4taatacgact cactataggg
aaataagaga gaaaagaaga gtaagaagaa atataagagc 60caccatgctc tctaacctca
ggattctgct caacaacgct gctctgcgga aaggccatac 120ctctgtcgtc
aggcacttct ggtgtgggaa acccgtgcag agccaggtgc agctcaaggg
180gcgggacctg ctcaccctga aaaatttcac aggcgaggaa atcaagtaca
tgctctggct 240gtctgccgat ctgaagttca ggatcaagca gaagggcgaa
tatctcccac tgctccaggg 300gaaaagtctg ggtatgatct tcgaaaagcg
gagtactagg accagactgt caacagagac 360tggattcgct ctgctcggag
gacacccatg ctttctgacc acacaggaca ttcatctcgg 420tgtgaacgag
tcactgaccg acacagctcg agtcctcagc tccatggcag atgccgtgct
480ggcaagggtc tacaaacaga gtgacctcga taccctggct aaggaagcaa
gcatccccat 540cattaatgga ctctccgacc tgtatcaccc tatccagatt
ctggccgatt acctcaccct 600gcaggagcat tattctagtc tgaaagggct
cacactgagc tggattggcg acggaaacaa 660tatcctgcac tccattatga
tgtctgccgc taagtttggc atgcatctgc aggcagccac 720accaaaagga
tacgaacccg atgcttccgt gactaagctg gccgaacagt atgctaaaga
780gaacggaact aagctgctcc tgaccaatga ccccctggag gctgcacacg
ggggtaacgt 840cctgatcact gatacctgga tttccatggg ccaggaggaa
gagaagaaaa agcgcctgca 900ggcattccag ggataccagg tgacaatgaa
aactgccaag gtcgccgctt ctgattggac 960ttttctccat tgtctgcccc
gaaagcctga agaggtggac gatgaggtct tctattcacc 1020tcggagcctg
gtgtttccag aagccgagaa tcgcaagtgg acaatcatgg cagtgatggt
1080gtccctcctc acagactatt ccccacagct ccagaagccc aagttttgag
cggccgctta 1140attaagctgc cttctgcggg gcttgccttc tggccatgcc
cttcttctct cccttgcacc 1200tgtacctctt ggtctttgaa taaagcctga
gtaggaagtc tagagtttaa acatttaaat 1260ct 126251262DNAArtificialcDNA
encoding human ornithine transcarbamylase, codon-optimized for
human expressionpromoter(1)..(20)T7 promoter 5taatacgact cactataggg
aaataagaga gaaaagaaga gtaagaagaa atataagagc 60caccatgctg tttaacctga
ggattctgct gaacaacgct gcttttcgga acggccacaa 120ctttatggtg
cggaactttc ggtgcggaca gccactgcag aacaaagtgc agctgaaggg
180gagggacctg ctgaccctga aaaatttcac aggagaggaa atcaagtaca
tgctgtggct 240gtctgccgat ctgaagttcc ggatcaagca gaagggcgaa
tatctgccac tgctgcaggg 300caaaagtctg gggatgatct tcgaaaagag
gagtactcgg accagactgt caacagagac 360tggattcgct ctgctgggag
gacacccatg ctttctgacc acacaggaca ttcatctggg 420cgtgaacgag
tcactgaccg acacagctcg agtcctgagc tccatggcag atgccgtgct
480ggcacgggtc tacaaacaga gcgacctgga taccctggct aaggaagcaa
gcatccccat 540cattaatggg ctgtccgacc tgtatcaccc tatccagatt
ctggccgatt acctgaccct 600gcaggagcat tattctagtc tgaaaggcct
gacactgagc tggattgggg acggaaacaa 660tatcctgcac tccattatga
tgtctgccgc taagtttgga atgcatctgc aggcagccac 720accaaaaggc
tacgaacccg atgccagtgt gactaagctg gccgaacagt atgctaaaga
780gaacggcact aagctgctgc tgaccaatga ccctctggag gctgcacacg
gaggcaacgt 840cctgatcact gatacctgga tttccatggg ccaggaggaa
gagaagaaaa agcgcctgca 900ggcattccag gggtaccagg tgacaatgaa
aactgccaag gtcgccgctt ctgattggac 960ttttctgcat tgtctgcccc
gaaaacctga agaggtggac gatgaggtct tctattcacc 1020taggagcctg
gtgtttccag aagccgagaa tcgcaagtgg acaatcatgg ctgtgatggt
1080gtccctgctg actgattatt ccccccagct gcagaaacct aagttctgag
cggccgctta 1140attaagctgc cttctgcggg gcttgccttc tggccatgcc
cttcttctct cccttgcacc 1200tgtacctctt ggtctttgaa taaagcctga
gtaggaagtc tagagtttaa acatttaaat 1260ct 126261221RNAArtificialmRNA
encoding human ornithine transcarbamylase, codon-optimized for
mouse expression 6gggaaauaag agagaaaaga agaguaagaa gaaauauaag
agccaccaug cuguucaacc 60ucagaauccu ccucaauaac gccgccuuua gaaacgguca
uaacuucaug gucagaaacu 120uuagaugugg ucagccucuc cagaacaaag
ugcagcucaa ggggcgggac cugcucaccc 180ugaaaaauuu cacaggcgag
gaaaucaagu acaugcucug gcugucugcc gaucugaagu 240ucaggaucaa
gcagaagggc gaauaucucc cacugcucca ggggaaaagu cuggguauga
300ucuucgaaaa gcggaguacu aggaccagac ugucaacaga gacuggauuc
gcucugcucg 360gaggacaccc augcuuucug accacacagg acauucaucu
cggugugaac gagucacuga 420ccgacacagc ucgaguccuc agcuccaugg
cagaugccgu gcuggcaagg gucuacaaac 480agagugaccu cgauacccug
gcuaaggaag caagcauccc caucauuaau ggacucuccg 540accuguauca
cccuauccag auucuggccg auuaccucac ccugcaggag cauuauucua
600gucugaaagg gcucacacug agcuggauug gcgacggaaa caauauccug
cacuccauua 660ugaugucugc cgcuaaguuu ggcaugcauc ugcaggcagc
cacaccaaaa ggauacgaac 720ccgaugcuuc cgugacuaag cuggccgaac
aguaugcuaa agagaacgga acuaagcugc 780uccugaccaa ugacccccug
gaggcugcac acggggguaa cguccugauc acugauaccu 840ggauuuccau
gggccaggag gaagagaaga aaaagcgccu gcaggcauuc cagggauacc
900aggugacaau gaaaacugcc aaggucgccg cuucugauug gacuuuucuc
cauugucugc 960cccgaaagcc ugaagaggug gacgaugagg ucuucuauuc
accucggagc cugguguuuc 1020cagaagccga gaaucgcaag uggacaauca
uggcagugau ggugucccuc cucacagacu 1080auuccccaca gcuccagaag
cccaaguuuu gagcggccgc uuaauuaagc ugccuucugc 1140ggggcuugcc
uucuggccau gcccuucuuc ucucccuugc accuguaccu cuuggucuuu
1200gaauaaagcc ugaguaggaa g 122171221RNAArtificialmRNA encoding
human ornithine transcarbamylase with mouse mitochondrial leader
sequence, codon-optimized for mouse expression 7gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug cucucuaacc 60ucaggauucu
gcucaacaac gcugcucugc ggaaaggcca uaccucuguc gucaggcacu
120ucuggugugg gaaacccgug cagagccagg ugcagcucaa ggggcgggac
cugcucaccc 180ugaaaaauuu cacaggcgag gaaaucaagu acaugcucug
gcugucugcc gaucugaagu 240ucaggaucaa gcagaagggc gaauaucucc
cacugcucca ggggaaaagu cuggguauga 300ucuucgaaaa gcggaguacu
aggaccagac ugucaacaga gacuggauuc gcucugcucg 360gaggacaccc
augcuuucug accacacagg acauucaucu cggugugaac gagucacuga
420ccgacacagc ucgaguccuc agcuccaugg cagaugccgu gcuggcaagg
gucuacaaac 480agagugaccu cgauacccug gcuaaggaag caagcauccc
caucauuaau ggacucuccg 540accuguauca cccuauccag auucuggccg
auuaccucac ccugcaggag cauuauucua 600gucugaaagg gcucacacug
agcuggauug gcgacggaaa caauauccug cacuccauua 660ugaugucugc
cgcuaaguuu ggcaugcauc ugcaggcagc cacaccaaaa ggauacgaac
720ccgaugcuuc cgugacuaag cuggccgaac aguaugcuaa agagaacgga
acuaagcugc 780uccugaccaa ugacccccug gaggcugcac acggggguaa
cguccugauc acugauaccu 840ggauuuccau gggccaggag gaagagaaga
aaaagcgccu gcaggcauuc cagggauacc 900aggugacaau gaaaacugcc
aaggucgccg cuucugauug gacuuuucuc cauugucugc 960cccgaaagcc
ugaagaggug gacgaugagg ucuucuauuc accucggagc cugguguuuc
1020cagaagccga gaaucgcaag uggacaauca uggcagugau ggugucccuc
cucacagacu 1080auuccccaca gcuccagaag cccaaguuuu gagcggccgc
uuaauuaagc ugccuucugc 1140ggggcuugcc uucuggccau gcccuucuuc
ucucccuugc accuguaccu cuuggucuuu 1200gaauaaagcc ugaguaggaa g
122181221RNAArtificialmRNA encoding human ornithine
transcarbamylase, codon-optimized for human expression 8gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug cuguuuaacc 60ugaggauucu
gcugaacaac gcugcuuuuc ggaacggcca caacuuuaug gugcggaacu
120uucggugcgg acagccacug cagaacaaag ugcagcugaa ggggagggac
cugcugaccc 180ugaaaaauuu cacaggagag gaaaucaagu acaugcugug
gcugucugcc gaucugaagu 240uccggaucaa gcagaagggc gaauaucugc
cacugcugca gggcaaaagu cuggggauga 300ucuucgaaaa gaggaguacu
cggaccagac ugucaacaga gacuggauuc gcucugcugg 360gaggacaccc
augcuuucug accacacagg acauucaucu gggcgugaac gagucacuga
420ccgacacagc ucgaguccug agcuccaugg cagaugccgu gcuggcacgg
gucuacaaac 480agagcgaccu ggauacccug gcuaaggaag caagcauccc
caucauuaau gggcuguccg 540accuguauca cccuauccag auucuggccg
auuaccugac ccugcaggag cauuauucua 600gucugaaagg ccugacacug
agcuggauug gggacggaaa caauauccug cacuccauua 660ugaugucugc
cgcuaaguuu ggaaugcauc ugcaggcagc cacaccaaaa ggcuacgaac
720ccgaugccag ugugacuaag cuggccgaac aguaugcuaa agagaacggc
acuaagcugc 780ugcugaccaa ugacccucug gaggcugcac acggaggcaa
cguccugauc acugauaccu 840ggauuuccau gggccaggag gaagagaaga
aaaagcgccu gcaggcauuc cagggguacc 900aggugacaau gaaaacugcc
aaggucgccg cuucugauug gacuuuucug cauugucugc 960cccgaaaacc
ugaagaggug gacgaugagg ucuucuauuc accuaggagc cugguguuuc
1020cagaagccga gaaucgcaag uggacaauca uggcugugau ggugucccug
cugacugauu 1080auucccccca gcugcagaaa ccuaaguucu gagcggccgc
uuaauuaagc ugccuucugc 1140ggggcuugcc uucuggccau gcccuucuuc
ucucccuugc accuguaccu cuuggucuuu 1200gaauaaagcc ugaguaggaa g
12219750PRTHomo sapiensTRANSIT(1)..(32)native mitochondrial leader
sequence 9Met Leu Arg Ala Lys Asn Gln Leu Phe Leu Leu Ser Pro His
Tyr Leu 1 5 10 15 Arg Gln Val Lys Glu Ser Ser Gly Ser Arg Leu Ile
Gln Gln Arg Leu 20 25 30 Leu His Gln Gln Gln Pro Leu His Pro Glu
Trp Ala Ala Leu Ala Lys 35 40 45 Lys Gln Leu Lys Gly Lys Asn Pro
Glu Asp Leu Ile Trp His Thr Pro 50 55 60 Glu Gly Ile Ser Ile Lys
Pro Leu Tyr Ser Lys Arg Asp Thr Met Asp 65 70 75 80 Leu Pro Glu Glu
Leu Pro Gly Val Lys Pro Phe Thr Arg Gly Pro Tyr 85 90 95 Pro Thr
Met Tyr Thr Phe Arg Pro Trp Thr Ile Arg Gln Tyr Ala Gly 100 105 110
Phe Ser Thr Val Glu Glu Ser Asn Lys Phe Tyr Lys Asp Asn Ile Lys 115
120 125 Ala Gly Gln Gln Gly Leu Ser Val Ala Phe Asp Leu Ala Thr His
Arg 130 135 140 Gly Tyr Asp Ser Asp Asn Pro Arg Val Arg Gly Asp Val
Gly Met Ala 145 150 155 160 Gly Val Ala Ile Asp Thr Val Glu Asp Thr
Lys Ile Leu Phe Asp Gly 165 170 175 Ile Pro Leu Glu Lys Met Ser Val
Ser Met Thr Met Asn Gly Ala Val 180 185 190 Ile Pro Val Leu Ala Asn
Phe Ile Val Thr Gly Glu Glu Gln Gly Val 195 200 205 Pro Lys Glu Lys
Leu Thr Gly Thr Ile Gln Asn Asp Ile Leu Lys Glu 210 215 220 Phe Met
Val Arg Asn Thr Tyr Ile Phe Pro Pro Glu Pro Ser Met Lys 225 230 235
240 Ile Ile Ala Asp Ile Phe Glu Tyr Thr Ala Lys His Met Pro Lys Phe
245 250
255 Asn Ser Ile Ser Ile Ser Gly Tyr His Met Gln Glu Ala Gly Ala Asp
260 265 270 Ala Ile Leu Glu Leu Ala Tyr Thr Leu Ala Asp Gly Leu Glu
Tyr Ser 275 280 285 Arg Thr Gly Leu Gln Ala Gly Leu Thr Ile Asp Glu
Phe Ala Pro Arg 290 295 300 Leu Ser Phe Phe Trp Gly Ile Gly Met Asn
Phe Tyr Met Glu Ile Ala 305 310 315 320 Lys Met Arg Ala Gly Arg Arg
Leu Trp Ala His Leu Ile Glu Lys Met 325 330 335 Phe Gln Pro Lys Asn
Ser Lys Ser Leu Leu Leu Arg Ala His Cys Gln 340 345 350 Thr Ser Gly
Trp Ser Leu Thr Glu Gln Asp Pro Tyr Asn Asn Ile Val 355 360 365 Arg
Thr Ala Ile Glu Ala Met Ala Ala Val Phe Gly Gly Thr Gln Ser 370 375
380 Leu His Thr Asn Ser Phe Asp Glu Ala Leu Gly Leu Pro Thr Val Lys
385 390 395 400 Ser Ala Arg Ile Ala Arg Asn Thr Gln Ile Ile Ile Gln
Glu Glu Ser 405 410 415 Gly Ile Pro Lys Val Ala Asp Pro Trp Gly Gly
Ser Tyr Met Met Glu 420 425 430 Cys Leu Thr Asn Asp Val Tyr Asp Ala
Ala Leu Lys Leu Ile Asn Glu 435 440 445 Ile Glu Glu Met Gly Gly Met
Ala Lys Ala Val Ala Glu Gly Ile Pro 450 455 460 Lys Leu Arg Ile Glu
Glu Cys Ala Ala Arg Arg Gln Ala Arg Ile Asp 465 470 475 480 Ser Gly
Ser Glu Val Ile Val Gly Val Asn Lys Tyr Gln Leu Glu Lys 485 490 495
Glu Asp Ala Val Glu Val Leu Ala Ile Asp Asn Thr Ser Val Arg Asn 500
505 510 Arg Gln Ile Glu Lys Leu Lys Lys Ile Lys Ser Ser Arg Asp Gln
Ala 515 520 525 Leu Ala Glu Arg Cys Leu Ala Ala Leu Thr Glu Cys Ala
Ala Ser Gly 530 535 540 Asp Gly Asn Ile Leu Ala Leu Ala Val Asp Ala
Ser Arg Ala Arg Cys 545 550 555 560 Thr Val Gly Glu Ile Thr Asp Ala
Leu Lys Lys Val Phe Gly Glu His 565 570 575 Lys Ala Asn Asp Arg Met
Val Ser Gly Ala Tyr Arg Gln Glu Phe Gly 580 585 590 Glu Ser Lys Glu
Ile Thr Ser Ala Ile Lys Arg Val His Lys Phe Met 595 600 605 Glu Arg
Glu Gly Arg Arg Pro Arg Leu Leu Val Ala Lys Met Gly Gln 610 615 620
Asp Gly His Asp Arg Gly Ala Lys Val Ile Ala Thr Gly Phe Ala Asp 625
630 635 640 Leu Gly Phe Asp Val Asp Ile Gly Pro Leu Phe Gln Thr Pro
Arg Glu 645 650 655 Val Ala Gln Gln Ala Val Asp Ala Asp Val His Ala
Val Gly Ile Ser 660 665 670 Thr Leu Ala Ala Gly His Lys Thr Leu Val
Pro Glu Leu Ile Lys Glu 675 680 685 Leu Asn Ser Leu Gly Arg Pro Asp
Ile Leu Val Met Cys Gly Gly Val 690 695 700 Ile Pro Pro Gln Asp Tyr
Glu Phe Leu Phe Glu Val Gly Val Ser Asn 705 710 715 720 Val Phe Gly
Pro Gly Thr Arg Ile Pro Lys Ala Ala Val Gln Val Leu 725 730 735 Asp
Asp Ile Glu Lys Cys Leu Glu Lys Lys Gln Gln Ser Val 740 745 750
102409RNAArtificialmRNA encoding human methylmalonyl-coenzyme A
mutase 10gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaug
uuaagagcua 60agaaucagcu uuuuuuacuu ucaccucauu accugaggca gguaaaagaa
ucaucaggcu 120ccaggcucau acagcaacga cuucuacacc agcaacagcc
ccuucaccca gaaugggcug 180cccuggcuaa aaagcagcug aaaggcaaaa
acccagaaga ccuaauaugg cacaccccgg 240aagggaucuc uauaaaaccc
uuguauucca agagagauac uauggacuua ccugaagaac 300uuccaggagu
gaagccauuc acacguggac cauauccuac cauguauacc uuuaggcccu
360ggaccauccg ccaguaugcu gguuuuagua cuguggaaga aagcaauaag
uucuauaagg 420acaacauuaa ggcuggucag cagggauuau caguugccuu
ugaucuggcg acacaucgug 480gcuaugauuc agacaacccu cgaguucgug
gugauguugg aauggcugga guugcuauug 540acacugugga agauaccaaa
auucuuuuug auggaauucc uuuagaaaaa augucaguuu 600ccaugacuau
gaauggagca guuauuccag uucuugcaaa uuuuauagua acuggagaag
660aacaaggugu accuaaagag aagcuuacug guaccaucca aaaugauaua
cuaaaggaau 720uuaugguucg aaauacauac auuuuuccuc cagaaccauc
caugaaaauu auugcugaca 780uauuugaaua uacagcaaag cacaugccaa
aauuuaauuc aauuucaauu aguggauacc 840auaugcagga agcaggggcu
gaugccauuc uggagcuggc cuauacuuua gcagauggau 900uggaguacuc
uagaacugga cuccaggcug gccugacaau ugaugaauuu gcaccaaggu
960ugucuuucuu cuggggaauu ggaaugaauu ucuauaugga aauagcaaag
augagagcug 1020guagaagacu cugggcucac uuaauagaga aaauguuuca
gccuaaaaac ucaaaaucuc 1080uucuucuaag agcacacugu cagacaucug
gauggucacu uacugagcag gaucccuaca 1140auaauauugu ccguacugca
auagaagcaa uggcagcagu auuuggaggg acucagucuu 1200ugcacacaaa
uucuuuugau gaagcuuugg guuugccaac ugugaaaagu gcucgaauug
1260ccaggaacac acaaaucauc auucaagaag aaucugggau ucccaaagug
gcugauccuu 1320ggggagguuc uuacaugaug gaaugucuca caaaugaugu
uuaugaugcu gcuuuaaagc 1380ucauuaauga aauugaagaa auggguggaa
uggccaaagc uguagcugag ggaauaccua 1440aacuucgaau ugaagaaugu
gcugcccgaa gacaagcuag aauagauucu gguucugaag 1500uaauuguugg
aguaaauaag uaccaguugg aaaaagaaga cgcuguagaa guucuggcaa
1560uugauaauac uucagugcga aacaggcaga uugaaaaacu uaagaagauc
aaauccagca 1620gggaucaagc uuuggcugaa cguugucuug cugcacuaac
cgaaugugcu gcuagcggag 1680auggaaauau ccuggcucuu gcaguggaug
caucucgggc aagauguaca gugggagaaa 1740ucacagaugc ccugaaaaag
guauuuggug aacauaaagc gaaugaucga auggugagug 1800gagcauaucg
ccaggaauuu ggagaaagua aagagauaac aucugcuauc aagaggguuc
1860auaaauucau ggaacgugaa ggucgcagac cucgucuucu uguagcaaaa
augggacaag 1920auggccauga cagaggagca aaaguuauug cuacaggauu
ugcugaucuu gguuuugaug 1980uggacauagg cccucuuuuc cagacuccuc
gugaaguggc ccagcaggcu guggaugcgg 2040augugcaugc ugugggcaua
agcacccucg cugcugguca uaaaacccua guuccugaac 2100ucaucaaaga
acuuaacucc cuuggacggc cagauauucu ugucaugugu ggagggguga
2160uaccaccuca ggauuaugaa uuucuguuug aaguuggugu uuccaaugua
uuugguccug 2220ggacucgaau uccaaaggcu gccguucagg ugcuugauga
uauugagaag uguuuggaaa 2280agaagcagca aucuguauaa gcggccgcuu
aauuaagcug ccuucugcgg ggcuugccuu 2340cuggccaugc ccuucuucuc
ucccuugcac cuguaccucu uggucuuuga auaaagccug 2400aguaggaag
240911728PRTHomo sapiensTRANSIT(1)..(52)native mitochondrial leader
sequence 11Met Ala Gly Phe Trp Val Gly Thr Ala Pro Leu Val Ala Ala
Gly Arg 1 5 10 15 Arg Gly Arg Trp Pro Pro Gln Gln Leu Met Leu Ser
Ala Ala Leu Arg 20 25 30 Thr Leu Lys His Val Leu Tyr Tyr Ser Arg
Gln Cys Leu Met Val Ser 35 40 45 Arg Asn Leu Gly Ser Val Gly Tyr
Asp Pro Asn Glu Lys Thr Phe Asp 50 55 60 Lys Ile Leu Val Ala Asn
Arg Gly Glu Ile Ala Cys Arg Val Ile Arg 65 70 75 80 Thr Cys Lys Lys
Met Gly Ile Lys Thr Val Ala Ile His Ser Asp Val 85 90 95 Asp Ala
Ser Ser Val His Val Lys Met Ala Asp Glu Ala Val Cys Val 100 105 110
Gly Pro Ala Pro Thr Ser Lys Ser Tyr Leu Asn Met Asp Ala Ile Met 115
120 125 Glu Ala Ile Lys Lys Thr Arg Ala Gln Ala Val His Pro Gly Tyr
Gly 130 135 140 Phe Leu Ser Glu Asn Lys Glu Phe Ala Arg Cys Leu Ala
Ala Glu Asp 145 150 155 160 Val Val Phe Ile Gly Pro Asp Thr His Ala
Ile Gln Ala Met Gly Asp 165 170 175 Lys Ile Glu Ser Lys Leu Leu Ala
Lys Lys Ala Glu Val Asn Thr Ile 180 185 190 Pro Gly Phe Asp Gly Val
Val Lys Asp Ala Glu Glu Ala Val Arg Ile 195 200 205 Ala Arg Glu Ile
Gly Tyr Pro Val Met Ile Lys Ala Ser Ala Gly Gly 210 215 220 Gly Gly
Lys Gly Met Arg Ile Ala Trp Asp Asp Glu Glu Thr Arg Asp 225 230 235
240 Gly Phe Arg Leu Ser Ser Gln Glu Ala Ala Ser Ser Phe Gly Asp Asp
245 250 255 Arg Leu Leu Ile Glu Lys Phe Ile Asp Asn Pro Arg His Ile
Glu Ile 260 265 270 Gln Val Leu Gly Asp Lys His Gly Asn Ala Leu Trp
Leu Asn Glu Arg 275 280 285 Glu Cys Ser Ile Gln Arg Arg Asn Gln Lys
Val Val Glu Glu Ala Pro 290 295 300 Ser Ile Phe Leu Asp Ala Glu Thr
Arg Arg Ala Met Gly Glu Gln Ala 305 310 315 320 Val Ala Leu Ala Arg
Ala Val Lys Tyr Ser Ser Ala Gly Thr Val Glu 325 330 335 Phe Leu Val
Asp Ser Lys Lys Asn Phe Tyr Phe Leu Glu Met Asn Thr 340 345 350 Arg
Leu Gln Val Glu His Pro Val Thr Glu Cys Ile Thr Gly Leu Asp 355 360
365 Leu Val Gln Glu Met Ile Arg Val Ala Lys Gly Tyr Pro Leu Arg His
370 375 380 Lys Gln Ala Asp Ile Arg Ile Asn Gly Trp Ala Val Glu Cys
Arg Val 385 390 395 400 Tyr Ala Glu Asp Pro Tyr Lys Ser Phe Gly Leu
Pro Ser Ile Gly Arg 405 410 415 Leu Ser Gln Tyr Gln Glu Pro Leu His
Leu Pro Gly Val Arg Val Asp 420 425 430 Ser Gly Ile Gln Pro Gly Ser
Asp Ile Ser Ile Tyr Tyr Asp Pro Met 435 440 445 Ile Ser Lys Leu Ile
Thr Tyr Gly Ser Asp Arg Thr Glu Ala Leu Lys 450 455 460 Arg Met Ala
Asp Ala Leu Asp Asn Tyr Val Ile Arg Gly Val Thr His 465 470 475 480
Asn Ile Ala Leu Leu Arg Glu Val Ile Ile Asn Ser Arg Phe Val Lys 485
490 495 Gly Asp Ile Ser Thr Lys Phe Leu Ser Asp Val Tyr Pro Asp Gly
Phe 500 505 510 Lys Gly His Met Leu Thr Lys Ser Glu Lys Asn Gln Leu
Leu Ala Ile 515 520 525 Ala Ser Ser Leu Phe Val Ala Phe Gln Leu Arg
Ala Gln His Phe Gln 530 535 540 Glu Asn Ser Arg Met Pro Val Ile Lys
Pro Asp Ile Ala Asn Trp Glu 545 550 555 560 Leu Ser Val Lys Leu His
Asp Lys Val His Thr Val Val Ala Ser Asn 565 570 575 Asn Gly Ser Val
Phe Ser Val Glu Val Asp Gly Ser Lys Leu Asn Val 580 585 590 Thr Ser
Thr Trp Asn Leu Ala Ser Pro Leu Leu Ser Val Ser Val Asp 595 600 605
Gly Thr Gln Arg Thr Val Gln Cys Leu Ser Arg Glu Ala Gly Gly Asn 610
615 620 Met Ser Ile Gln Phe Leu Gly Thr Val Tyr Lys Val Asn Ile Leu
Thr 625 630 635 640 Arg Leu Ala Ala Glu Leu Asn Lys Phe Met Leu Glu
Lys Val Thr Glu 645 650 655 Asp Thr Ser Ser Val Leu Arg Ser Pro Met
Pro Gly Val Val Val Ala 660 665 670 Val Ser Val Lys Pro Gly Asp Ala
Val Ala Glu Gly Gln Glu Ile Cys 675 680 685 Val Ile Glu Ala Met Lys
Met Gln Asn Ser Met Thr Ala Gly Lys Thr 690 695 700 Gly Thr Val Lys
Ser Val His Cys Gln Ala Gly Asp Thr Val Gly Glu 705 710 715 720 Gly
Asp Leu Leu Val Glu Leu Glu 725 122343RNAArtificialmRNA encoding
human propionyl CoA carboxylase, alpha polypeptide (PCCA)
12gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaug gcgggguucu
60gggucgggac agcaccgcug gucgcugccg gacggcgugg gcgguggccg ccgcagcagc
120ugaugcugag cgcggcgcug cggacccuga agcauguucu guacuauuca
agacagugcu 180uaaugguguc ccguaaucuu gguucagugg gauaugaucc
uaaugaaaaa acuuuugaua 240aaauucuugu ugcuaauaga ggagaaauug
caugucgggu uauuagaacu ugcaagaaga 300ugggcauuaa gacaguugcc
auccacagug auguugaugc uaguucuguu caugugaaaa 360uggcggauga
ggcugucugu guuggcccag cucccaccag uaaaagcuac cucaacaugg
420augccaucau ggaagccauu aagaaaacca gggcccaagc uguacaucca
gguuauggau 480uccuuucaga aaacaaagaa uuugccagau guuuggcagc
agaagauguc guuuucauug 540gaccugacac acaugcuauu caagccaugg
gcgacaagau ugaaagcaaa uuauuagcua 600agaaagcaga gguuaauaca
aucccuggcu uugauggagu agucaaggau gcagaagaag 660cugucagaau
ugcaagggaa auuggcuacc cugucaugau caaggccuca gcagguggug
720gugggaaagg caugcgcauu gcuugggaug augaagagac cagggauggu
uuuagauugu 780caucucaaga agcugcuucu aguuuuggcg augauagacu
acuaauagaa aaauuuauug 840auaauccucg ucauauagaa auccagguuc
uaggugauaa acaugggaau gcuuuauggc 900uuaaugaaag agagugcuca
auucagagaa gaaaucagaa ggugguggag gaagcaccaa 960gcauuuuuuu
ggaugcggag acucgaagag cgaugggaga acaagcugua gcucuugcca
1020gagcaguaaa auauuccucu gcugggaccg uggaguuccu uguggacucu
aagaagaauu 1080uuuauuucuu ggaaaugaau acaagacucc agguugagca
uccugucaca gaaugcauua 1140cuggccugga ccuaguccag gaaaugaucc
guguugcuaa gggcuacccu cucaggcaca 1200aacaagcuga uauucgcauc
aacggcuggg caguugaaug ucggguuuau gcugaggacc 1260ccuacaaguc
uuuugguuua ccaucuauug ggagauuguc ucaguaccaa gaaccguuac
1320aucuaccugg uguccgagug gacaguggca uccaaccagg aagugauauu
agcauuuauu 1380augauccuau gauuucaaaa cuaaucacau auggcucuga
uagaacugag gcacugaaga 1440gaauggcaga ugcacuggau aacuauguua
uucgaggugu uacacauaau auugcauuac 1500uucgagaggu gauaaucaac
ucacgcuuug uaaaaggaga caucagcacu aaauuucucu 1560ccgaugugua
uccugauggc uucaaaggac acaugcuaac caagagugag aagaaccagu
1620uauuggcaau agcaucauca uuguuugugg cauuccaguu aagagcacaa
cauuuucaag 1680aaaauucaag aaugccuguu auuaaaccag acauagccaa
cugggagcuc ucaguaaaau 1740ugcaugauaa aguucauacc guaguagcau
caaacaaugg gucaguguuc ucgguggaag 1800uugauggguc gaaacuaaau
gugaccagca cguggaaccu ggcuucgccc uuauugucug 1860ucagcguuga
uggcacucag aggacugucc agugucuuuc ucgagaagca gguggaaaca
1920ugagcauuca guuucuuggu acaguguaca aggugaauau cuuaaccaga
cuugccgcag 1980aauugaacaa auuuaugcug gaaaaaguga cugaggacac
aagcaguguu cugcguuccc 2040cgaugcccgg agugguggug gccgucucug
ucaagccugg agacgcggua gcagaagguc 2100aagaaauuug ugugauugaa
gccaugaaaa ugcagaauag uaugacagcu gggaaaacug 2160gcacggugaa
aucugugcac ugucaagcug gagacacagu uggagaaggg gaucugcucg
2220uggagcugga augagcggcc gcuuaauuaa gcugccuucu gcggggcuug
ccuucuggcc 2280augcccuucu ucucucccuu gcaccuguac cucuuggucu
uugaauaaag ccugaguagg 2340aag 234313539PRTHomo
sapiensTRANSIT(1)..(28)native mitochondrial leader sequence 13Met
Ala Ala Ala Leu Arg Val Ala Ala Val Gly Ala Arg Leu Ser Val 1 5 10
15 Leu Ala Ser Gly Leu Arg Ala Ala Val Arg Ser Leu Cys Ser Gln Ala
20 25 30 Thr Ser Val Asn Glu Arg Ile Glu Asn Lys Arg Arg Thr Ala
Leu Leu 35 40 45 Gly Gly Gly Gln Arg Arg Ile Asp Ala Gln His Lys
Arg Gly Lys Leu 50 55 60 Thr Ala Arg Glu Arg Ile Ser Leu Leu Leu
Asp Pro Gly Ser Phe Val 65 70 75 80 Glu Ser Asp Met Phe Val Glu His
Arg Cys Ala Asp Phe Gly Met Ala 85 90 95 Ala Asp Lys Asn Lys Phe
Pro Gly Asp Ser Val Val Thr Gly Arg Gly 100 105 110 Arg Ile Asn Gly
Arg Leu Val Tyr Val Phe Ser Gln Asp Phe Thr Val 115 120 125 Phe Gly
Gly Ser Leu Ser Gly Ala His Ala Gln Lys Ile Cys Lys Ile 130 135 140
Met Asp Gln Ala Ile Thr Val Gly Ala Pro Val Ile Gly Leu Asn Asp 145
150 155 160 Ser Gly Gly Ala Arg Ile Gln Glu Gly Val Glu Ser Leu Ala
Gly Tyr 165 170 175 Ala Asp Ile Phe Leu Arg Asn Val Thr Ala Ser Gly
Val Ile Pro Gln 180 185 190 Ile Ser Leu Ile Met Gly Pro Cys Ala Gly
Gly Ala Val Tyr Ser Pro 195 200 205 Ala Leu Thr Asp Phe Thr Phe Met
Val Lys Asp Thr Ser Tyr Leu Phe 210 215 220 Ile Thr Gly Pro Asp Val
Val Lys Ser Val Thr Asn Glu Asp Val Thr 225 230 235 240 Gln Glu Glu
Leu Gly Gly Ala Lys Thr His Thr Thr Met Ser Gly Val 245 250 255 Ala
His Arg Ala Phe Glu Asn Asp Val Asp Ala Leu Cys Asn Leu Arg 260 265
270 Asp Phe Phe Asn Tyr Leu Pro Leu Ser Ser Gln Asp Pro Ala Pro Val
275 280 285 Arg Glu Cys His Asp Pro Ser Asp Arg Leu Val Pro Glu
Leu
Asp Thr 290 295 300 Ile Val Pro Leu Glu Ser Thr Lys Ala Tyr Asn Met
Val Asp Ile Ile 305 310 315 320 His Ser Val Val Asp Glu Arg Glu Phe
Phe Glu Ile Met Pro Asn Tyr 325 330 335 Ala Lys Asn Ile Ile Val Gly
Phe Ala Arg Met Asn Gly Arg Thr Val 340 345 350 Gly Ile Val Gly Asn
Gln Pro Lys Val Ala Ser Gly Cys Leu Asp Ile 355 360 365 Asn Ser Ser
Val Lys Gly Ala Arg Phe Val Arg Phe Cys Asp Ala Phe 370 375 380 Asn
Ile Pro Leu Ile Thr Phe Val Asp Val Pro Gly Phe Leu Pro Gly 385 390
395 400 Thr Ala Gln Glu Tyr Gly Gly Ile Ile Arg His Gly Ala Lys Leu
Leu 405 410 415 Tyr Ala Phe Ala Glu Ala Thr Val Pro Lys Val Thr Val
Ile Thr Arg 420 425 430 Lys Ala Tyr Gly Gly Ala Tyr Asp Val Met Ser
Ser Lys His Leu Cys 435 440 445 Gly Asp Thr Asn Tyr Ala Trp Pro Thr
Ala Glu Ile Ala Val Met Gly 450 455 460 Ala Lys Gly Ala Val Glu Ile
Ile Phe Lys Gly His Glu Asn Val Glu 465 470 475 480 Ala Ala Gln Ala
Glu Tyr Ile Glu Lys Phe Ala Asn Pro Phe Pro Ala 485 490 495 Ala Val
Arg Gly Phe Val Asp Asp Ile Ile Gln Pro Ser Ser Thr Arg 500 505 510
Ala Arg Ile Cys Cys Asp Leu Asp Val Leu Ala Ser Lys Lys Val Gln 515
520 525 Arg Pro Trp Arg Lys His Ala Asn Ile Pro Leu 530 535
141776RNAArtificialmRNA encoding human propionyl CoA carboxylase,
beta polypeptide (PCCB) 14gggaaauaag agagaaaaga agaguaagaa
gaaauauaag agccaccaug gcggcggcau 60uacggguggc ggcggucggg gcaaggcuca
gcguucuggc gagcggucuc cgcgccgcgg 120uccgcagccu uugcagccag
gccaccucug uuaacgaacg caucgaaaac aagcgccgga 180ccgcgcugcu
gggagggggc caacgccgua uugacgcgca gcacaagcga ggaaagcuaa
240cagccaggga gaggaucagu cucuugcugg acccuggcag cuuuguugag
agcgacaugu 300uuguggaaca cagaugugca gauuuuggaa uggcugcuga
uaagaauaag uuuccuggag 360acagcguggu cacuggacga ggccgaauca
auggaagauu gguuuauguc uucagucagg 420auuuuacagu uuuuggaggc
agucugucag gagcacaugc ccaaaagauc ugcaaaauca 480uggaccaggc
cauaacggug ggggcuccag ugauugggcu gaaugacucu gggggagcac
540ggauccaaga aggaguggag ucuuuggcug gcuaugcaga caucuuucug
aggaauguua 600cggcauccgg agucaucccu cagauuucuc ugaucauggg
cccaugugcu gguggggccg 660ucuacucccc agcccuaaca gacuucacgu
ucaugguaaa ggacaccucc uaccuguuca 720ucacuggccc ugauguugug
aagucuguca ccaaugagga uguuacccag gaggagcucg 780guggugccaa
gacccacacc accaugucag guguggccca cagagcuuuu gaaaaugaug
840uugaugccuu guguaaucuc cgggauuucu ucaacuaccu gccccugagc
agucaggacc 900cggcucccgu ccgugagugc cacgauccca gugaccgucu
gguuccugag cuugacacaa 960uugucccuuu ggaaucaacc aaagccuaca
acauggugga caucauacac ucuguuguug 1020augagcguga auuuuuugag
aucaugccca auuaugccaa gaacaucauu guugguuuug 1080caagaaugaa
ugggaggacu guuggaauug uuggcaacca accuaaggug gccucaggau
1140gcuuggauau uaauucaucu gugaaagggg cucguuuugu cagauucugu
gaugcauuca 1200auauuccacu caucacuuuu guugaugucc cuggcuuucu
accuggcaca gcacaggaau 1260acgggggcau cauccggcau ggugccaagc
uucucuacgc auuugcugag gcaacuguac 1320ccaaagucac agucaucacc
aggaaggccu auggaggugc cuaugauguc augagcucua 1380agcaccuuug
uggugauacc aacuaugccu ggcccaccgc agagauugca gucaugggag
1440caaagggcgc uguggagauc aucuucaaag ggcaugagaa uguggaagcu
gcucaggcag 1500aguacaucga gaaguuugcc aacccuuucc cugcagcagu
gcgaggguuu guggaugaca 1560ucauccaacc uucuuccaca cgugcccgaa
ucugcuguga ccuggauguc uuggccagca 1620agaagguaca acguccuugg
agaaaacaug caaauauucc auuguaagcg gccgcuuaau 1680uaagcugccu
ucugcggggc uugccuucug gccaugcccu ucuucucucc cuugcaccug
1740uaccucuugg ucuuugaaua aagccugagu aggaag
17761530PRTArtificialGALA peptide 15Trp Glu Ala Ala Leu Ala Glu Ala
Leu Ala Glu Ala Leu Ala Glu His 1 5 10 15 Leu Ala Glu Ala Leu Ala
Glu Ala Leu Glu Ala Leu Ala Ala 20 25 30 1618PRTArtificialtruncated
GALA peptide 16Cys Ala Glu Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu
Ala Glu Ala 1 5 10 15 Leu Ala 1726PRTApis mellifera 17Gly Ile Gly
Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu 1 5 10 15 Ile
Ser Trp Ile Lys Arg Lys Arg Gln Gln 20 25 1827PRTArtificialMelittin
peptide with N-terminal cysteine 18Cys Gly Ile Gly Ala Val Leu Lys
Val Leu Thr Thr Gly Leu Pro Ala 1 5 10 15 Leu Ile Ser Trp Ile Lys
Arg Lys Arg Gln Gln 20 25 1921PRTArtificialHPH-1 peptide 19Phe Ile
Ile Asp Ile Ile Ala Phe Leu Leu Met Gly Gly Phe Ile Val 1 5 10 15
Tyr Val Lys Asn Leu 20 2024PRTArtificialHPH-1 peptide with
N-terminal Cys-Ala-Ala 20Cys Ala Ala Phe Ile Ile Asp Ile Ile Ala
Phe Leu Leu Met Gly Gly 1 5 10 15 Phe Ile Val Tyr Val Lys Asn Leu
20 2117PRTArtificialsHGP peptide 21Cys Ala Arg Gly Trp Glu Val Leu
Lys Tyr Trp Trp Asn Leu Leu Gln 1 5 10 15 Tyr
2230PRTArtificialbPrPp peptide 22Met Val Lys Ser Lys Ile Gly Ser
Trp Ile Leu Val Leu Phe Val Ala 1 5 10 15 Met Trp Ser Asp Val Gly
Leu Cys Lys Lys Arg Pro Lys Pro 20 25 30 2318PRTArtificialMAP
peptide 23Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala Ala
Leu Lys 1 5 10 15 Leu Ala 2411PRTArtificialPTD4 peptide 24Tyr Ala
Arg Ala Ala Ala Arg Gln Ala Arg Ala 1 5 10
2529PRTArtificialMaurocalcine peptide 25Gly Asp Cys Leu Pro His Leu
Lys Leu Cys Lys Glu Asn Lys Asp Cys 1 5 10 15 Cys Ser Lys Lys Cys
Lys Arg Arg Gly Thr Asn Ile Glu 20 25 2610PRTArtificialSynB3
peptide 26Arg Arg Leu Ser Tyr Ser Arg Arg Arg Phe 1 5 10
2718PRTArtificialSynB1 peptide 27Arg Gly Gly Arg Leu Ser Tyr Ser
Arg Arg Arg Phe Ser Thr Ser Thr 1 5 10 15 Gly Arg
2817PRTArtificialYTA4 peptide 28Ile Ala Trp Val Lys Ala Phe Ile Arg
Lys Leu Arg Lys Gly Pro Leu 1 5 10 15 Gly 2916PRTArtificialYTA2
peptide 29Tyr Thr Ala Ile Ala Trp Val Lys Ala Phe Ile Arg Lys Leu
Arg Lys 1 5 10 15 3020PRTArtificialCADY peptide 30Gly Leu Trp Arg
Ala Leu Trp Arg Leu Leu Arg Ser Leu Trp Arg Leu 1 5 10 15 Leu Trp
Arg Ala 20 3115PRTArtificialPep-3 peptide 31Lys Trp Phe Glu Thr Trp
Phe Thr Glu Trp Pro Lys Lys Arg Lys 1 5 10 15
3221PRTArtificialPep-1 peptide 32Lys Glu Thr Trp Trp Glu Thr Trp
Trp Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20
3321PRTArtificialPepFect peptide (amide linkage from epsilon amino
group of Lys7) 33Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu Lys Ala
Leu Ala Ala Leu 1 5 10 15 Ala Lys Lys Ile Leu 20
3421PRTArtificialPepFect-3 peptide 34Ala Gly Tyr Leu Leu Gly Lys
Ile Asn Leu Lys Ala Leu Ala Ala Leu 1 5 10 15 Ala Lys Lys Ile Leu
20 3516PRTArtificialPenetratin peptide 35Arg Gln Ile Lys Ile Trp
Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
3630PRTArtificialKALA peptide 36Trp Glu Ala Lys Leu Ala Lys Ala Leu
Ala Lys Ala Leu Ala Lys His 1 5 10 15 Leu Ala Lys Ala Leu Ala Lys
Ala Leu Lys Ala Cys Glu Ala 20 25 30 3718PRTArtificialpVEC peptide
37Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His 1
5 10 15 Ser Lys 3829PRTArtificialRVG peptide 38Tyr Thr Ile Trp Met
Pro Glu Asn Pro Arg Pro Gly Thr Pro Cys Asp 1 5 10 15 Ile Phe Thr
Asn Ser Arg Gly Lys Arg Ala Ser Asn Gly 20 25 3916PRTArtificialMPS
peptide 39Ala Ala Val Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu
Ala Lys 1 5 10 15 4027PRTArtificialTransportan peptide 40Gly Trp
Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly Lys Ile Asn Leu 1 5 10 15
Lys Ala Leu Ala Ala Leu Ala Lys Lys Ile Leu 20 25
4112PRTArtificialTAT peptide 41Gly Arg Lys Lys Arg Arg Gln Arg Arg
Pro Pro Gln 1 5 10 4220PRTArtificialBMV Gag-(7-25) peptide 42Lys
Met Thr Arg Ala Gln Arg Arg Ala Ala Ala Arg Arg Asn Arg Arg 1 5 10
15 Trp Thr Ala Arg 20 4328PRTArtificialhCT(18-32)-k7 peptide
(branched structure between Ala13 and Lys14) 43Lys Lys Arg Lys Ala
Pro Lys Lys Lys Arg Lys Phe Ala Lys Phe His 1 5 10 15 Thr Phe Pro
Gln Thr Ala Ile Gly Val Gly Ala Pro 20 25 4422PRTArtificialM1073
peptide 44Met Val Thr Val Leu Phe Arg Arg Leu Arg Ile Arg Arg Ala
Ser Gly 1 5 10 15 Pro Pro Arg Val Arg Val 20 4523PRTArtificialEB1
peptide 45Leu Ile Arg Leu Trp Ser His Leu Ile His Ile Trp Phe Gln
Asn Arg 1 5 10 15 Arg Leu Lys Trp Lys Lys Lys 20
4627PRTArtificialMPG-beta peptide 46Gly Ala Leu Phe Leu Gly Phe Leu
Gly Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys
Lys Lys Arg Lys Val 20 25 4727PRTArtificialMPG-beta peptide 47Gly
Ala Leu Phe Leu Ala Phe Leu Ala Ala Ala Leu Ser Leu Met Gly 1 5 10
15 Leu Trp Ser Gln Pro Lys Lys Lys Arg Lys Val 20 25 48464PRTHomo
sapiens 48Met Ala Ser Glu Ser Gly Lys Leu Trp Gly Gly Arg Phe Val
Gly Ala 1 5 10 15 Val Asp Pro Ile Met Glu Lys Phe Asn Ala Ser Ile
Ala Tyr Asp Arg 20 25 30 His Leu Trp Glu Val Asp Val Gln Gly Ser
Lys Ala Tyr Ser Arg Gly 35 40 45 Leu Glu Lys Ala Gly Leu Leu Thr
Lys Ala Glu Met Asp Gln Ile Leu 50 55 60 His Gly Leu Asp Lys Val
Ala Glu Glu Trp Ala Gln Gly Thr Phe Lys 65 70 75 80 Leu Asn Ser Asn
Asp Glu Asp Ile His Thr Ala Asn Glu Arg Arg Leu 85 90 95 Lys Glu
Leu Ile Gly Ala Thr Ala Gly Lys Leu His Thr Gly Arg Ser 100 105 110
Arg Asn Asp Gln Val Val Thr Asp Leu Arg Leu Trp Met Arg Gln Thr 115
120 125 Cys Ser Thr Leu Ser Gly Leu Leu Trp Glu Leu Ile Arg Thr Met
Val 130 135 140 Asp Arg Ala Glu Ala Glu Arg Asp Val Leu Phe Pro Gly
Tyr Thr His 145 150 155 160 Leu Gln Arg Ala Gln Pro Ile Arg Trp Ser
His Trp Ile Leu Ser His 165 170 175 Ala Val Ala Leu Thr Arg Asp Ser
Glu Arg Leu Leu Glu Val Arg Lys 180 185 190 Arg Ile Asn Val Leu Pro
Leu Gly Ser Gly Ala Ile Ala Gly Asn Pro 195 200 205 Leu Gly Val Asp
Arg Glu Leu Leu Arg Ala Glu Leu Asn Phe Gly Ala 210 215 220 Ile Thr
Leu Asn Ser Met Asp Ala Thr Ser Glu Arg Asp Phe Val Ala 225 230 235
240 Glu Phe Leu Phe Trp Ala Ser Leu Cys Met Thr His Leu Ser Arg Met
245 250 255 Ala Glu Asp Leu Ile Leu Tyr Cys Thr Lys Glu Phe Ser Phe
Val Gln 260 265 270 Leu Ser Asp Ala Tyr Ser Thr Gly Ser Ser Leu Met
Pro Gln Lys Lys 275 280 285 Asn Pro Asp Ser Leu Glu Leu Ile Arg Ser
Lys Ala Gly Arg Val Phe 290 295 300 Gly Arg Cys Ala Gly Leu Leu Met
Thr Leu Lys Gly Leu Pro Ser Thr 305 310 315 320 Tyr Asn Lys Asp Leu
Gln Glu Asp Lys Glu Ala Val Phe Glu Val Ser 325 330 335 Asp Thr Met
Ser Ala Val Leu Gln Val Ala Thr Gly Val Ile Ser Thr 340 345 350 Leu
Gln Ile His Gln Glu Asn Met Gly Gln Ala Leu Ser Pro Asp Met 355 360
365 Leu Ala Thr Asp Leu Ala Tyr Tyr Leu Val Arg Lys Gly Met Pro Phe
370 375 380 Arg Gln Ala His Glu Ala Ser Gly Lys Ala Val Phe Met Ala
Glu Thr 385 390 395 400 Lys Gly Val Ala Leu Asn Gln Leu Ser Leu Gln
Glu Leu Gln Thr Ile 405 410 415 Ser Pro Leu Phe Ser Gly Asp Val Ile
Cys Val Trp Asp Tyr Gly His 420 425 430 Ser Val Glu Gln Tyr Gly Ala
Leu Gly Gly Thr Ala Arg Ser Ser Val 435 440 445 Asp Trp Gln Ile Arg
Gln Val Arg Ala Leu Leu Gln Ala Gln Gln Ala 450 455 460
491551RNAArtificialmRNA encoding human argininosuccinate lyase
(ASL) isoform 1, codon-optimized for mouse expression 49gggaaauaag
agagaaaaga agaguaagaa gaaauauaag agccaccaug gcaucagaga 60gcgguaaacu
gugggguggg agauucgugg gugccgucga uccuauuaug gagaaauuca
120acgccagcau ugccuacgac agacaccugu gggaggugga cguccagggc
ucaaaggccu 180acagccgggg ucuggagaag gcaggccugc ucacaaaagc
cgaaauggac cagauccugc 240acggacucga uaagguggcu gaggaauggg
cacaggggac auucaaacug aacucuaacg 300acgaggauau ccacacugcu
aacgagagga gacugaagga acucauuggc gccacagcug 360gaaaacugca
uacuggacgg agccgcaacg accagguggu cacagaucug agacucugga
420ugcggcagac cugcucuaca cugaguggac ugcucuggga gcucauucga
acuauggugg 480acagggcaga ggccgaaaga gacguccugu uuccaggaua
uacccaccug cagcgagcac 540agccaaucag guggucucac uggauucuga
gucacgcugu ggcacucacc cgcgauucug 600agcgacugcu cgaagugcga
aagaggauca acguccugcc ucucgggagu ggugccauug 660cugggaaucc
acugggugug gacagggagc ugcucagagc ugaacugaac uucggcgcaa
720ucacccugaa uucaauggac gccacaagcg agcgcgauuu ugucgccgaa
uuccucuuuu 780gggcuagucu gugcaugacc caucucucaa ggauggcuga
ggaccugauc cucuacugua 840caaaggaauu cagcuuugug cagcuguccg
acgcauauuc uacugguagc ucccugaugc 900cccagaagaa aaacccugac
ucccuggagc ucauuagauc uaaggcagga cgaguguucg 960gaaggugcgc
agggcugcuc augacucuga aaggccuccc auccaccuac aauaaggacc
1020ugcaggagga uaaagaagcc guguuugaag ucagugacac aaugucagcu
gugcugcagg 1080ucgcaacugg ugugaucagc acccugcaga uucaccagga
aaacauggga caggcucugu 1140ccccagacau gcuggccacu gaucucgcuu
acuaucuggu gcgaaaggga augccuuuca 1200ggcaggcaca cgaggccagc
ggcaaggcag uguuuauggc cgaaaccaaa ggcgucgccc 1260ugaaucagcu
gucccuccag gagcugcaga caaucagccc ccucuucucc ggggacguga
1320uuugugucug ggauuacgga cacucugugg aacaguacgg ggcccugggc
ggaaccgcua 1380gaagcagcgu cgauuggcag auuaggcagg uccgagcccu
ccuccaggca cagcaggccu 1440gagcggccgc uuaauuaagc ugccuucugc
ggggcuugcc uucuggccau gcccuucuuc 1500ucucccuugc accuguaccu
cuuggucuuu gaauaaagcc ugaguaggaa g 155150412PRTHomo sapiens 50Met
Ser Ser Lys Gly Ser Val Val Leu Ala Tyr Ser Gly Gly Leu Asp 1 5 10
15 Thr Ser Cys Ile Leu Val Trp Leu Lys Glu Gln Gly Tyr Asp Val Ile
20 25 30 Ala Tyr Leu Ala Asn Ile Gly Gln Lys Glu Asp Phe Glu Glu
Ala Arg 35 40 45 Lys Lys Ala Leu Lys Leu Gly Ala Lys Lys Val Phe
Ile Glu Asp Val 50 55 60 Ser Arg Glu Phe Val Glu Glu Phe Ile Trp
Pro Ala Ile Gln Ser Ser 65 70 75 80 Ala Leu Tyr Glu Asp Arg Tyr Leu
Leu Gly Thr Ser Leu Ala Arg Pro 85 90 95 Cys Ile Ala Arg Lys Gln
Val Glu Ile Ala Gln Arg Glu Gly Ala Lys 100 105 110 Tyr Val Ser His
Gly Ala Thr Gly Lys Gly Asn Asp Gln Val Arg Phe 115 120 125 Glu Leu
Ser Cys Tyr Ser Leu Ala Pro Gln Ile Lys Val Ile Ala Pro 130 135 140
Trp Arg Met Pro Glu Phe Tyr Asn Arg Phe Lys Gly Arg Asn Asp Leu 145
150 155 160 Met Glu Tyr Ala Lys Gln His Gly Ile Pro Ile
Pro Val Thr Pro Lys 165 170 175 Asn Pro Trp Ser Met Asp Glu Asn Leu
Met His Ile Ser Tyr Glu Ala 180 185 190 Gly Ile Leu Glu Asn Pro Lys
Asn Gln Ala Pro Pro Gly Leu Tyr Thr 195 200 205 Lys Thr Gln Asp Pro
Ala Lys Ala Pro Asn Thr Pro Asp Ile Leu Glu 210 215 220 Ile Glu Phe
Lys Lys Gly Val Pro Val Lys Val Thr Asn Val Lys Asp 225 230 235 240
Gly Thr Thr His Gln Thr Ser Leu Glu Leu Phe Met Tyr Leu Asn Glu 245
250 255 Val Ala Gly Lys His Gly Val Gly Arg Ile Asp Ile Val Glu Asn
Arg 260 265 270 Phe Ile Gly Met Lys Ser Arg Gly Ile Tyr Glu Thr Pro
Ala Gly Thr 275 280 285 Ile Leu Tyr His Ala His Leu Asp Ile Glu Ala
Phe Thr Met Asp Arg 290 295 300 Glu Val Arg Lys Ile Lys Gln Gly Leu
Gly Leu Lys Phe Ala Glu Leu 305 310 315 320 Val Tyr Thr Gly Phe Trp
His Ser Pro Glu Cys Glu Phe Val Arg His 325 330 335 Cys Ile Ala Lys
Ser Gln Glu Arg Val Glu Gly Lys Val Gln Val Ser 340 345 350 Val Leu
Lys Gly Gln Val Tyr Ile Leu Gly Arg Glu Ser Pro Leu Ser 355 360 365
Leu Tyr Asn Glu Glu Leu Val Ser Met Asn Val Gln Gly Asp Tyr Glu 370
375 380 Pro Thr Asp Ala Thr Gly Phe Ile Asn Ile Asn Ser Leu Arg Leu
Lys 385 390 395 400 Glu Tyr His Arg Leu Gln Ser Lys Val Thr Ala Lys
405 410 511395RNAArtificialmRNA encoding human argininosuccinate
synthetase (ASS1), codon-optimized for mouse expression
51gggaaauaag agagaaaaga agaguaagaa gaaauauaag agccaccaug agcucaaagg
60ggaguguggu gcuggccuau ucuggcgggc uggauaccuc uugcauucug guguggcuga
120aggaacaggg uuacgacgug aucgcauacc uggccaacau ugggcagaag
gaggauuuug 180aggaagcuag aaagaaagca cugaaacucg gcgccaagaa
aguguucauc gaggacgucu 240cccgggaauu cguggaggaa uuuaucuggc
cagccauuca gagcuccgcu cuguacgagg 300auagauaucu gcucggaacc
agccucgcac gacccugcau cgccaggaag cagguggaga 360uugcucagcg
cgaaggggca aaguacgucu cccacggugc cacaggcaaa ggaaacgacc
420aggugcgauu ugagcugucu uguuauaguc ucgcacccca gaucaagguc
auugcccccu 480ggcgcaugcc cgaguucuac aaccgguuua agggccgcaa
cgaccugaug gaauacgcua 540aacagcacgg aaucccaauu cccgugacuc
cuaagaaccc cugguccaug gaugagaauc 600ugaugcauau cucuuacgag
gcugggauuc ucgaaaaccc uaagaaucag gcacccccug 660gucuguauac
uaagacccag gacccagcca aagcucccaa cacaccugau auccuggaga
720uugaauuuaa gaaaggggug ccugucaaag ugacuaacgu gaaagacggu
accacacacc 780agaccucucu ggagcucuuu auguaccuga acgaagucgc
aggcaagcac ggggugggua 840gaaucgauau ugucgagaau cgguucaucg
ggaugaaaag ucgcgguauu uacgaaaccc 900cugcuggaac aauccuguau
cacgcccauc ucgacauuga ggcuuucaca auggauagag 960aagugagaaa
gaucaaacag ggccugggac ucaaguucgc cgagcuggug uacacuggau
1020uuuggcacuc uccagagugc gaauucgugc gacauuguau cgcuaagagu
caggagaggg 1080ucgaagggaa gguccaggug ucaguccuga aaggccaggu
guacauucuc ggacgggagu 1140caccccugag ccucuauaac gaggaacugg
ugagcaugaa cguccagggc gacuacgaac 1200cuacagacgc cacuggauuc
aucaacauca acucacucag gcucaaggaa uaccacaggc 1260uccagucaaa
agucacagca aaguaggcgg ccgcuuaauu aagcugccuu cugcggggcu
1320ugccuucugg ccaugcccuu cuucucuccc uugcaccugu accucuuggu
cuuugaauaa 1380agccugagua ggaag 1395
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